bluuebunny/crossref_metadata_2025 huggingface dataset:
Thyroid Storm in a Child following Radioactive Iodine (RAI) Therapy: A Consequence of RAI Versus Withdrawal of Antithyroid Medication
Authors: Kadmon Penny M. , Noto Richard B. , Boney Charlotte M. , Goodwin Gregory , Gruppuso Philip A.
Published: January 2014

A 7.5-yr-old boy with Graves’ disease, difficult to control with antithyroid medication and radioactive iodine (RAI) therapy, developed thyroid storm encephalopathy on day 13 after withdrawal of methimazole therapy, 4 days after iodione-131 treatment. We attributed his thyroid storm to withdrawal of antithyroid medication as opposed to RAI therapy. We interpret this case as indicating that there may be a need to reevaluate the duration of antithyroid medication withdrawal before RAI therapy for hyperthyroid children at increased risk for thyroid storm.
The Effect of Aromatase Inhibition on Sex Steroids, Gonadotropins, and Markers of Bone Turnover in Older Men1
Authors: Taxel Pamela , Kennedy Dayna G. , Fall Pamela M. , Willard Alice K. , Clive Jonathan M. , Raisz Lawrence G.
Published: January 2014

There is evidence that estrogen decreases bone turnover in men as well as women. We therefore hypothesized that older men would show increased bone resorption in response to inhibition of the aromatase enzyme, which converts androgens to estrogen.

Fifteen eugonadal men over 65 yr were treated for 9 weeks with 2.0 mg/day of anastrozole, an aromatase inhibitor. After 9 weeks of treatment, there were significant decreases in estradiol, estrone, and sex hormone-binding globulin levels by 29%, 73%, and 16%, respectively, and total testosterone increased significantly by 56%. Despite the limited decrease of estrogen and the increase in testosterone, C-telopeptide of type 1 collagen showed a progressive significant increase of 11%, 24%, and 33% (P for trend = 0.033) above baseline at 3, 6, and 9 weeks, respectively. N-telopeptide of type 1 collagen values were highly correlated with C-telopeptide of type 1 collagen, but the change in N-telopeptide of type 1 collagen was not statistically significant. Bone-specific alkaline phosphatase and N-terminal type I procollagen peptides showed significant decreases of 8% and 11% of baseline at 9 weeks. Osteocalcin decreased significantly by 30% at 18 weeks.

We conclude that aromatase inhibition can reduce estrogen levels in older men, but this effect is limited, perhaps because of feedback stimulation of testosterone production, and that endogenous estrogen derived from aromatization of testosterone plays a role in bone metabolism of older men by limiting the rate of bone resorption.
Prolactin (PRL)-Releasing Peptide Stimulates PRL Secretion from Human Fetal Pituitary Cultures and Growth Hormone Release from Cultured Pituitary Adenomas1
Authors: Rubinek Tami , Hadani Moshe , Barkai Gad , Melmed Shlomo , Shimon Ilan
Published: January 2014

The hypothalamic peptide PRL-releasing peptide (PrRP) has recently been cloned and identified as a ligand of an orphan pituitary receptor that stimulates in vitro PRL secretion. PrRP also induces PRL release in rats in vivo, especially in normal cycling females. However, no information on the effects of PrRP in the human is available. To elucidate the role of PrRP in regulating human anterior pituitary hormones, we used human PrRP-31 in primary cultures of human pituitary tissues, including fetal (20–27 weeks gestation) and normal adult pituitaries, as well as PRL- and GH-secreting adenomas. PrRP increased PRL secretion from human fetal pituitary cultures in a dose-dependent manner by up to 35% (maximal effect achieved with 10 nm), whereas TRH was slightly more potent for PRL release. Coincubation with estradiol resulted in enhanced fetal PRL response to PrRP, and GH release was only increased in the presence of estradiol. Although PRL secretion from PRL-cell adenomas was not affected by PrRP, PrRP induced PRL release from cultures of a GH-cell adenoma that cosecreted PRL. PrRP enhanced GH release in several GH-secreting adenomas studied by 25–27%, including GH stimulation in a mixed PRL-GH-cell tumor. These results show for the first time direct in vitro effects of PrRP-31 on human pituitary cells. PrRP is less potent than TRH in releasing PRL from human fetal lactotrophs and is unable to release PRL from PRL-cell adenomas in culture, but stimulated GH from several somatotroph adenomas. Thus, PrRP may participate in regulating GH, in addition to PRL, in the human pituitary.
RAPID COMMUNICATION: Inhibitory Effect of Pioglitazone on Carotid Arterial Wall Thickness in Type 2 Diabetes
Authors: Koshiyama Hiroyuki , Shimono Dai , Kuwamura Naomitsu , Minamikawa Jun , Nakamura Yoshio
Published: January 2014

There have been increasing evidences that thiazolidinediones, peroxisome proliferator-activated receptor γ (PPARγ) agonists, may have some antiatherogenic actions. We have previously reported that troglitazone has a potent inhibitory effect on common carotid arterial intima-media thickness (IMT) in subjects with type 2 diabetes. However, some studies suggested a possibility that PPARγ activators may have protoatherogenic actions, raising concern about their detrimental effects in diabetic subjects. In the present study, we investigated the effect of treatment with pioglitazone, another PPARγ agonist, on IMT in a total of 106 Japanese subjects with type 2 diabetes. Pioglitazone (30 mg daily) was administered for 6 months in 53 patients. Compared to control group (n = 53), the group given pioglitazone showed a significant decrease in IMT as early as 3 months after the administration. The decrease in IMT was also found after 6 months (IMT change: −0.084[SE 0.023] mm vs. control 0.022[SE 0.006] mm, P < 0.001), although the difference between those after 3 and 6 months did not reach any statistical significance. These findings indicate that thiazolidinediones cause an inhibition of early atherosclerotic process PPARγ activation.
Successful Long-Term Treatment of Refractory Cushing’s Disease with High-Dose Mifepristone (RU 486)
Authors: Chu James W. , Matthias Dwight F. , Belanoff Joseph , Schatzberg Alan , Hoffman Andrew R. , Feldman David
Published: January 2014

An extremely ill patient, with Cushing’s syndrome caused by an ACTH-secreting pituitary macroadenoma, experienced complications of end-stage cardiomyopathy, profound psychosis, and multiple metabolic disturbances. Initially treated unsuccessfully by a combination of conventional surgical, medical, and radiotherapeutic approaches, he responded dramatically to high-dose long-term mifepristone therapy (up to 25 mg/kg·d). Treatment efficacy was confirmed by the normalization of all biochemical glucocorticoid-sensitive measurements, as well as by the significant reversal of the patient’s heart failure, the resolution of his psychotic depression, and the eventual unusual return of his adrenal axis to normal. His 18-month-long mifepristone treatment course was notable for development of severe hypokalemia that was attributed to excessive cortisol activation of the mineralocorticoid receptor, which responded to spironolactone administration. This case illustrates the efficacy of high-dose long-term treatment with mifepristone in refractory Cushing’s syndrome. The case also demonstrates the potential need for concomitant mineralocorticoid receptor blockade in mifepristone-treated Cushing’s disease, because cortisol levels may rise markedly, reflecting corticotroph disinhibition, to cause manifestations of mineralocorticoid excess.
Insulin Resistance as a Predictor of Age-Related Diseases
Authors: Facchini Francesco S. , Hua Nancy , Abbasi Fahim , Reaven Gerald M.
Published: January 2014

The current study was initiated to evaluate the ability of insulin resistance to predict a variety of age-related diseases. Baseline measurements of insulin resistance and related variables were made between 1988–1995 in 208 apparently healthy, nonobese (body mass index < 30 kg/m2) individuals, who were then evaluated 4–11 yr later (mean ± sem = 6.3 ± 0.2 yr) for the appearance of the following age-related diseases: hypertension, coronary heart disease, stroke, cancer, and type 2 diabetes. The effect of insulin resistance on the development of clinical events was evaluated by dividing the study group into tertiles of insulin resistance at baseline and comparing the events in these 3 groups. Clinical endpoints (n = 40) were identified in 37 individuals (18%) of those evaluated, including 12 with hypertension, 3 with hypertension + type 2 diabetes, 9 with cancer, 7 with coronary heart disease, 4 with stroke, and 2 with type 2 diabetes. Twenty-eight out of the total 40 clinical events were seen in 25 individuals (36%) in the most insulin-resistant tertile, with the other 12 occurring in the group with an intermediate degree of insulin resistance. Furthermore, insulin resistance was an independent predictor of all clinical events, using both multiple logistic regression and Cox’s proportional hazards analysis. The fact that an age-related clinical event developed in approximately 1 out of 3 healthy individuals in the upper tertile of insulin resistance at baseline, followed for an average of 6 yr, whereas no clinical events were observed in the most insulin-sensitive tertile, should serve as a strong stimulus to further efforts to define the role of insulin resistance in the genesis of age-related diseases.
Cross-Sectional and Prospective Associations between Abdominal Adiposity and Proinsulin Concentration
Authors: Hanley Anthony J. G. , McKeown-Eyssen Gail , Harris Stewart B. , Hegele Robert A. , Wolever Thomas M. S. , Kwan Jeremy , Zinman Bernard
Published: January 2014

The objective of this study was to investigate the associations of total and abdominal obesity with variation in proinsulin concentration in a Native Canadian population experiencing an epidemic of type 2 diabetes mellitus (DM).

Between 1993 and 1995, 728 members of a Native Canadian community participated in a population-based survey to determine the prevalence and risk factors for type 2 DM. Samples for glucose, C-peptide, and proinsulin were drawn after an overnight fast, and a 75-g oral glucose tolerance test was administered. Type 2 DM and impaired glucose tolerance (IGT) were diagnosed using World Health Organization criteria. Height, weight, waist circumference, and percent body fat were measured. In 1998, 95 individuals who, at baseline, had IGT or normal glucose tolerance with an elevated 2-h glucose level (≥7.0 mm) participated in a follow-up evaluation using the same protocol.

After adjustment for age, sex, C-peptide concentration, per cent body fat, and waist circumference, proinsulin was found to be significantly elevated in diabetic subjects, relative to subjects with both impaired and normal glucose tolerance (both P < 0.0001); and the concentration in those with IGT was higher, compared with normals (P < 0.0001). Among nondiabetic subjects, proinsulin showed significant univariate associations with percent body fat, body mass index, and waist circumference (r = 0.34, 0.45, 0.41, respectively, all P < 0.0001). After adjustment for body fat and other covariates, waist circumference remained significantly associated with proinsulin concentration in nondiabetic subjects (r = 0.20, P < 0.0001). In prospective analysis, adjusted for covariates (including baseline IGT and follow-up glucose tolerance status), baseline waist circumference was positively associated with both follow-up and change in proinsulin concentration (r = 0.27, P = 0.01; r = 0.24, P = 0.03, respectively).

These data highlight the detrimental effects of abdominal obesity on β-cell function, and support the hypothesis that β-cell dysfunction occurs early in the natural history of glucose intolerance.
Glial-Derived Neurotropic Factor and RET Gene Expression in Normal Human Anterior Pituitary Cell Types and in Pituitary Tumors
Authors: Japón Miguel A. , Urbano Angel G. , Sáez Carmen , Segura Dolores I. , Cerro Alfonso Leal , Diéguez Carlos , Alvarez Clara V.
Published: January 2014

Glial-derived neurotropic factor (GDNF) signaling is mediated through a 2-component system consisting of the so-called GDNF receptor-α (GFRα1), which binds to GDNF. This complex activates the tyrosine kinase receptor RET. In this paper we demonstrate GDNF, GFRα1, and RET mRNA and protein expression in the human anterior pituitary gland. Double immunohistochemistry of anterior pituitary sections showed GDNF immunoreactivity in more than 95% of somatotrophs and to a lesser extent in corticotrophs (20%); it was almost absent in the remaining cell types. Also, although more than 95% of somatotrophs were stained for RET, no positive immunostaining could be detected in other cell types. Furthermore, we have looked for GDNF and RET in human pituitary adenomas of various hormonal phenotypes. Strong positive immunostaining was found for c-RET in all of the GH-secreting adenomas screened as well as in 50% of ACTH-producing adenomas. Positive immunostaining for GDNF was found in all of the GH-secreting adenomas and in 10% of the corticotropinomas. Lastly, we found strong positive immunostaining for GFRα1 in 90% of the somatotropinomas and 50% of the corticotropinomas as well as in 1 of 8 prolactinomas and 1 of 13 nonfunctioning adenomas. All of the remaining pituitary tumors screened were negative for RET, GDNF, and GFRα1.

This study indicates that GDNF may well be acting in the regulation of somatotroph cell growth and/or cell function in the normal human anterior pituitary gland. The expression of RET in all of the somatotropinomas and in 50% of the ACTH-producing tumors implies that GDNF and RET could be involved in the pathogenesis of pituitary tumors.
Expression and Function of Vasoactive Intestinal Peptide, Pituitary Adenylate Cyclase-Activating Polypeptide, and Their Receptors in the Human Adrenal Gland
Authors: Mazzocchi G. , Malendowicz L. K. , Rebuffat P. , Gottardo L. , Nussdorfer Gastone G.
Published: January 2014

VIP and pituitary adenylate cyclase-activating polypeptide (PACAP) are two regulatory peptides that possess remarkable amino acid sequence homology and act through common receptors, named PAC1, VPAC1, and VPAC2. PAC1 receptor is selective for PACAP, whereas VPAC1 and VPAC2 receptors bind both VIP and PACAP. We have investigated the expression and function of VIP, PACAP, and their receptors in the zona glomerulosa (ZG), zonae fasciculata and reticularis, and adrenal medulla (AM) of the human adrenal cortex. RT-PCR and RIA detected VIP and PACAP expression exclusively in AM cells. RT-PCR demonstrated the presence of PAC1 mRNA only in AM and of VPAC1 and VPAC2 mRNAs in both ZG and AM cells. VIP and PACAP concentration-dependently increased aldosterone and catecholamine secretion from cultured ZG and AM cells. The catecholamine response to both peptides was higher than the aldosterone response, and the secretagogue action of PACAP was more intense than that of VIP. The aldosterone response of cultured ZG cells to VIP or PACAP was unaffected by the PAC1 receptor antagonist PACAP-(6–38) (PAC1-A), but was significantly decreased by the VPAC1 receptor antagonist [Ac-His1,d-Phe2,Lys15,Arg16]VIP-(3–7),GH-releasing factor-(8–27)-NH2 (VPAC1-A). The catecholamine response of cultured AM cells to VIP was lowered by VPAC1-A and unaffected by PAC1-A; conversely, the catecholamine response to PACAP was reduced by both PAC1-A and VPAC1-A. Simultaneous exposure to both antagonists did not abolish the catecholamine response to PACAP. Collectively, our findings allow us to conclude that in human adrenals 1) VIP and PACAP biosynthesis exclusively occurs in AM cells; 2) ZG cells are provided with functional VPAC1 and VPAC2 receptors, whose activation by VIP or PACAP elicits a moderate aldosterone response; 3) AM cells possess PAC1, VPAC1, and VPAC2 receptors, whose activation evokes a marked catecholamine response; and 4) the catecholamine response to PACAP is more intense than that to VIP, because it is mediated by all subtypes of VIP/PACAP receptors.
Genetic or Enzymatic Disruption of Aromatase Inhibits the Growth of Ectopic Uterine Tissue
Authors: Fang Zongjuan , Yang Sijun , Gurates Bilgin , Tamura Mitsutoshi , Simpson Evan , Evans Dean , Bulun Serdar E.
Published: January 2014

Aromatase P450 (P450arom) is the key enzyme for the biosynthesis of estrogen that is essential for the growth of human endometriosis, a pathology characterized by endometrium-like tissue on the peritoneal surfaces of abdominal organs manifest by pelvic pain and infertility. Surgically transplanted autologous uterine tissue to ectopic sites on the peritoneum in mice has been used as an animal model to study endometriosis. Using this mouse model, we evaluated the roles of the P450arom gene and aromatase enzyme activity in the growth of endometriosis represented by ectopic uterine tissues in mice. Endometriosis was induced surgically in the following groups of mice: 1) untreated transgenic mice with disrupted P450arom gene (ArKO); 2) ArKO mice treated with systemic estrogen; 3) untreated wild-type (WT) mice; 4) WT mice treated with estrogen; 5) WT mice treated with the aromatase inhibitor, letrozole; and 6) WT mice treated with letrozole and estrogen. Each group contained eight mice; +/+ littermates of ArKO mice were used as WT controls. Treatment with estrogen increased the size of ectopic uterine tissues in ArKO and WT mice significantly. The ectopic uterine lesions in untreated and estrogen-treated ArKO mice were strikingly smaller than those in untreated and estrogen-treated WT controls, respectively. Systemic treatment of WT mice with letrozole significantly decreased the lesion size in a dose-dependent manner. The addition of estrogen to letrozole treatment increased the ectopic lesion size, although these lesions were significantly smaller than those in mice treated with estrogen only. As tissue controls, the effects of these conditions on normally located (eutopic) uterine tissue were evaluated. The effects of disruption of the P450arom gene and treatments with letrozole and estrogen seemed to be more profound on ectopic tissues, suggesting that ectopic tissues might be more sensitive to estrogen for growth. We conclude that both an intact P450arom gene and the presence of aromatase enzyme activity are essential for the growth of ectopic uterine tissue in a mouse model of endometriosis.
Norepinephrine Spillover from Human Adipose Tissue before and after a 72-Hour Fast
Authors: Patel J. N. , Coppack S. W. , Goldstein D. S. , Miles J. M. , Eisenhofer G.
Published: January 2014

Adipose tissue lipolysis is at least in part stimulated by the sympathetic nervous system (SNS). Although there is a generalized decrease in SNS activity with fasting, the rate of lipolysis during fasting increases. The aim of this study was to determine whether there is an association between activation of sympathetic nerves innervating adipose tissue and the increase in lipolysis seen during fasting in humans. We used the isotope dilution technique to measure regional norepinephrine spillover from abdominal sc adipose tissue from seven healthy subjects before and after a 72-h fast. Our results showed a significant increase in adipose tissue spillover of norepinephrine (mean ± sem, 0.40 ± 0.09 vs. 1.08 ± 0.18 pmol·100 g−1·min−1, P < 0.05) and arterial norepinephrine concentrations (0.92 ± 0.10 vs. 1.23 ± 0.08 nmol·liter−1, P < 0.05) after the fast with no significant change in total body norepinephrine spillover, forearm norepinephrine spillover, epinephrine concentrations, or energy expenditure. We show for the first time, in humans, a selective regional increase in adipose tissue norepinephrine spillover in response to a 72-h fast and suggest that the SNS may play a greater role in the regulation of lipid metabolism during fasting than previously thought.
Inverse Regulation of Protein Turnover and Amino Acid Transport in Skeletal Muscle of Hypercatabolic Patients
Authors: Biolo Gianni , Fleming R. Y. Declan , Maggi Sergio P. , Nguyen Thuan T. , Herndon David N. , Wolfe Robert R.
Published: January 2014

We have investigated the relationships between the rates of muscle protein synthesis and degradation and of transmembrane transport of selected amino acids in leg skeletal muscle of 19 severely burned patients and 18 normal controls in the postabsorptive state. Patients were studied on the 14 ± 5 postburn day, and their mean burn size was 66% ± 18% of total body surface area. Methods were based on the leg arteriovenous balance technique in combination with biopsies of the vastus lateralis muscle and infusions of isotopic tracers of amino acids. Net muscle protein breakdown was greater in the patients because of an 83% increase in the rate of muscle protein degradation. The rate of muscle protein synthesis was also increased in the patients but to a lesser extent than protein degradation, i.e. by 50% with the arteriovenous phenylalanine balance technique and by 49% with the direct tracer incorporation method. The absolute values of inward transport of phenylalanine, leucine, and lysine were not significantly different in the two groups. However, the ability of transport systems to take up amino acids from the bloodstream, as assessed by dividing inward transport by amino acid delivery to leg muscle, were 50–63% lower in the patients. In contrast, outward phenylalanine and lysine transport were 40% and 67% greater in the patients than in the controls, respectively. We conclude the primary alteration in muscle protein metabolism is an acceleration of protein breakdown, and the increase in protein synthesis likely is due to increased intracellular amino acid availability as a result of accelerated breakdown. Transmembrane transport in the outward direction is accelerated, presumably to facilitate the export of amino acids from muscle to other tissues. In contrast, transmembrane transport in the inward direction is impaired relatively to the increased delivery of circulating amino acid to skeletal muscle secondary to accelerated blood flow.
RET Expression in Papillary Thyroid Cancer from Patients Irradiated in Childhood for Benign Conditions
Authors: Collins B. J. , Chiappetta G. , Schneider A. B. , Santoro M. , Pentimalli F. , Fogelfeld L. , Gierlowski T. , Shore-Freedman E. , Jaffe G. , Fusco A.
Published: January 2014

Both external and internal exposure to radiation have been linked to the development of papillary thyroid cancer. Rearrangement of the gene for RET tyrosine kinase and subsequent expression of this protein has also been found to occur in many papillary thyroid cancers, and with increased frequency in radiation-related cancers following the Chernobyl accident. However, little has been reported on the frequency of RET rearrangements in cancers after exposure to external radiation. We here report on RET protein immunoreactivity in paraffin-embedded thyroid samples from 30 patients with papillary thyroid cancer who received radiation treatment during childhood for benign conditions at Michael Reese Hospital in Chicago, and in 34 patients identified from the tumor registry as having papillary thyroid cancer with no history of therapeutic radiation. The subjects were characterized by sex, age at surgery, and the following attributes of tumor pathology: size, number of lobes involved, number of foci, lymph node metastases, and soft tissue invasion. Representative tissue samples were reacted with an antibody against the RET tyrosine kinase domain whose expression has been shown to correlate highly with RET/PTC rearrangements. A greater percentage of cancers positive for RET immunoreactivity was found in the radiation-exposed group (86.7% vs. 52.9%, P = 0.006). Although the mean age at surgery of the exposed group was lower than the control group, there was no correlation of positive RET immunoreactivity with the age at surgery. No characteristics of the tumors were associated with positive RET immunoreactivity. In summary, the greater incidence of RET-immunopositives in the irradiated group indicates that the expression of RET immunoreactivity is strongly associated with radiation exposure, but the prognostic significance of this is not yet clear.
Peripheral Serotonin Enhances Lipid Metabolism by Accelerating Bile Acid Turnover
Authors: Watanabe Hitoshi , Akasaka Daisuke , Ogasawara Hideki , Sato Kan , Miyake Masato , Saito Kazuki , Takahashi Yu , Kanaya Takashi , Takakura Ikuro , Hondo Tetsuya , Chao Guozheng , Rose Michael T. , Ohwada Shyuichi , Watanabe Kouichi , Yamaguchi Takahiro , Aso Hisashi
Published: January 2014

Endocrinology, published August 4, 2010, 10.1210/en.2009-1349
Identification of Amino Acids in the τ2-Region of the Mouse Glucocorticoid Receptor That Contribute to Hormone Binding and Transcriptional Activation
Authors: Milhon Jon , Lee Sunyoung , Kohli Kulwant , Chen Dagang , Hong Heng , Stallcup Michael R.
Published: January 2014

The τ2-region of steroid hormone receptors is a highly conserved region located at the extreme N-terminal end of the hormone-binding domain. A protein fragment encoding τ2 has been shown to function as an independent transcriptional activation domain; however, because this region is essential for hormone binding, it has been difficult to determine whether the τ2-region also contributes to the transactivation function of intact steroid receptors. In this study a series of amino acid substitutions were engineered at conserved positions in the τ2-region of the mouse glucocorticoid receptor (mGR, amino acids 533–562) to map specific amino acid residues that contribute to the hormone-binding function, transcriptional activation, or both. Substitution of alanine or glycine for some amino acids (mutations E546G, P547A, and D555A) reduced or eliminated hormone binding, but the transactivation function of the intact GR and/or the minimum τ2-fragment was unaffected for each of these mutants. Substitution of alanine for amino acid S561 reduced transactivation activity in the intact GR and the minimum τ2-fragment but had no effect on hormone binding. The single mutation L550A and the double amino acid substitution L541G+L542G affected both hormone binding and transactivation. The fact that the S561A and L550A substitutions each caused a loss of transactivation activity in the minimum τ2-fragment and the full-length GR indicated that the τ2-region does contribute to the overall transactivation function of the full-length GR. Overall, the N-terminal portion of the τ2-region (mGR 541–547) was primarily involved in hormone binding, whereas the C-terminal portion of theτ 2-region (mGR 548–561) was primarily involved in transactivation.
Stress-Induced Transcriptional Regulation in the Developing Rat Brain Involves Increased Cyclic Adenosine 3′,5′-Monophosphate-Regulatory Element Binding Activity
Authors: Hatalski Carolyn G. , Baram Tallie Z.
Published: January 2014

The cAMP-regulatory element (CRE) binding protein (CREB) functions as a trans-acting regulator of genes containing the CRE sequence in their promoter. These include a number of critical genes, such as CRF, involved in the hypothalamic response to stressful stimuli in the adult. The ability of the developing rat (during the first 2 postnatal weeks) to mount the full complement of this stress response has been questioned. We have previously demonstrated the stress-induced up-regulation of the transcription of hypothalamic CRF during the second postnatal week in the rat. The focus of the current study was to explore the mechanism of transcriptional regulation in response to stress through the physiological induction of transcriptional trans-activators that bind to the CRE in the developing rat brain. CRE-binding activity was detected via gel shift analysis in extracts from both the hypothalamus and the cerebral cortex of the developing rat. CREB was identified in these extracts by Western blot analysis and was shown to be the major contributor to the CRE-binding activity by gel shift analysis with two specific antibodies directed against CREB. After acute hypothermic stress, the abundance of CRE-binding activity (but not of total immunoreactive CREB), increased in hypothalamic extracts. This enhanced CRE-binding activity was blocked by an antiserum directed against CREB and was accompanied by an apparent increase in CREB phosphorylation. These results indicate that posttranslational enhancement of CRE-binding activity is likely to constitute an important mechanism for up-regulation of genes possessing the CRE sequence in the developing rat hypothalamus by adverse external signals.
Mapping the Domains of the Interaction of the Vitamin D Receptor and Steroid Receptor Coactivator-1
Authors: Gill Rajbir K. , Atkins Loretta M. , Hollis Bruce W. , Bell Norman H.
Published: January 2014

The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) and mediates the effects of the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3[ 1,25-(OH)2D3], on gene expression. The VDR binds to the VDRE as a heterodimeric complex with retinoid X receptor. In the present study, we have used a yeast two-hybrid system to clone complementary DNA that codes for VDR-interacting protein(s). We found that the human steroid receptor coactivator-1 (SRC-1) interacts with the VDR in a ligand-dependent manner, as demonstrated by β-galactosidase production. The interaction of the VDR and the SRC-1 takes place at physiological concentrations of 1,25(OH)2D3. A 48.2-fold stimulation of β-galactosidase activity was observed in the presence of 10−10m 1,25-(OH)2D3. In addition, a direct interaction between the ligand-activated glutathione-S-transferase-VDR and 35S-labeled SRC-1 was observed in vitro. Deletion-mutation analysis of the VDR established that the ligand-dependent activation domain (AF-2) of the VDR is required for the interaction with SRC-1. One deletion mutant, pGVDR-(1–418), bound the ligand but failed to interact with the SRC-1, whereas another deletion mutant, pGVDR-(1–423), bound the ligand and interacted with the SRC-1. We demonstrated that all the deletion mutants were expressed as analyzed by a Gal4 DNA-binding domain antibody. Deletion mutation analysis of the SRC-1 demonstrated that 27 amino acids (DPCNTNPTPMTKATPEEIKLEAQS-QFT) of the SRC-1 are essential for interaction with the AF-2 motif of the VDR.
Endothelin-1 Is a Potent Survival Factor for c-Myc-Dependent Apoptosis
Authors: Shichiri Masayoshi , Sedivy John M. , Marumo Fumiaki , Hirata Yukio
Published: January 2014

Many vertebrate cells are resistant to apoptotic stimuli, whose variety and the mechanisms involved are not fully understood. Endothelin-1 is an endothelium-derived vasoactive peptide that mediates many physiological functions, such as vasoconstriction and cell proliferation. Deregulated expression of c-Myc induces apoptosis in serum-deprived fibroblasts. Using a panel of isogenic fibroblast cell lines with differential c-myc expression levels, we demonstrate that low doses of endothelin-1 protect fibroblasts against serum deprivation-induced apoptosis, which occurs through a c-Myc-dependent process. The endothelin-1-induced cell survival was mediated by the ETA receptor and was not linked to the ability of endothelin-1 to induce cell proliferation. The survival function of endothelin-1 was abrogated by inhibiting the mitogen-activated protein kinase pathway. These results demonstrate a hitherto unappreciated role of endothelin-1 as a potent survival factor for c-Myc-dependent apoptosis, a process mediated by the ETA receptor and the mitogen-activated protein kinase pathway.
Androgen Responsiveness of Mouse Kidney β-Glucuronidase Requires 5′-Flanking and Intragenic Gus-s Sequences
Authors: Thornton S. , Thomas D. W. , Gallagher P. M. , Ganschow R. E.
Published: January 2014

Genetics studies of natural variants of the androgen response of mouse β-glucuronidase (GUS) reveal a cis-active element closely linked to the GUS structural gene (Gus-s) that is necessary for this kidney-specific response. Results of our previous studies suggested sequences within or near an androgen-inducible deoxyribonuclease I-hypersensitive site (DH site) located in the ninth intron of Gus-s are associated with the androgen response of GUS. Using transgenic mice, we now demonstrate that at least two regions of sequence within Gus-s are involved in regulating the androgen response of GUS. The first, located within 3.8 kb of Gus-s 5′-flanking sequence, directs the response and its tissue specificity, while the second, located within a 6.4-kb fragment of Gus-s extending from the third through the ninth intron of Gus-s, protects the androgen responsiveness of the transgene from repressive influences of the insertion site.
Truncated SNAP-25 (1–197), Like Botulinum Neurotoxin A, Can Inhibit Insulin Secretion from HIT-T15 Insulinoma Cells
Authors: Huang Xiaohang , Wheeler Michael B. , Kang You-hou , Sheu Laura , Lukacs Gergely L. , Trimble William S. , Gaisano Herbert Y.
Published: January 2014

We and others have previously shown that insulin-secreting cells of the pancreas express high levels of SNAP-25 (synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) implicated in synaptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin. To determine whether the inhibition of release was due to the depletion of functional SNAP-25 or the accumulation of proteolytic by-products, we transfected cells with SNAP-25 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin. This modified SNAP-25 (amino acids 1–197) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K+-mediated plasma membrane depolarization. Electron microscopy revealed that cells transfected with either BoNT/A or truncated SNAP-25 contained significantly higher numbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhibitory action of BoNT/A toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.
Regulation of the Rat Thyrotropin Receptor Gene by the Methylation-Sensitive Transcription Factor GA-Binding Protein
Authors: Yokomori Norihiko , Tawata Masato , Saito Tukasa , Shimura Hiroki , Onaya Toshimasa
Published: January 2014

The GA-binding protein (GABP), a transcription factor with a widespread tissue distribution, consists of two subunits,α and β1, and acts as a potent positive regulator of various genes. The effect of GABP on transcription of the TSH receptor (TSHR) gene in rat FRTL-5 thyroid cells has now been investigated. Both deoxyribonuclease I footprint analysis and gel mobility-shift assays indicated that bacterially expressed glutathione S-transferase fusion proteins of GABP subunits bind to a region spanning nucleotides (nt) −116 to −80 of the TSHR gene. In gel mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells yielded several specific bands with a probe comprising nt −116 to− 80. Supershift assays with antibodies to GABPα and to GABPβ1 showed that GABP was a component of the probe complexes formed by the nuclear extracts. Immunoblot analysis confirmed the presence of both GABP subunits in the nuclear extracts. A reporter gene construct containing the TSHR gene promoter was activated, in a dose-dependent manner, in FRTL-5 cells by cotransfection with constructs encoding both GABPα and GABPβ1. Both GABP binding to and activation of the TSHR gene promoter were prevented by methylation of CpG sites at nt −93 and− 85.

These CpG sites were highly methylated (>82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.
The Adrenomedullin Gene Is a Target for Negative Regulation by the Myc Transcription Complex
Authors: Wang Xueyan , Peters Mette A. , Utama Fransiscus E. , Wang Yuzhen , Taparowsky Elizabeth J.
Published: January 2014

The Myc family of transcription factors plays a central role in vertebrate growth and development although relatively few genetic targets of the Myc transcription complex have been identified. In this study, we used mRNA differential display to investigate gene expression changes induced by the overexpression of the MC29 v-Myc oncoprotein in C3H10T1/2 mouse fibroblasts. We identified the transcript of the adrenomedullin gene (AM) as an mRNA that is specifically down-regulated in v-Myc overexpressing C3H10T1/2 cell lines as well as in a Rat 1a cell line inducible for c-Myc. Nucleotide sequence analysis of the mouse AM promoter reveals the presence of consensus CAAT and TATA boxes as well as an initiator element (INR) with significant sequence similarity to the INR responsible for Myc-mediated repression of the adenovirus major late promoter (AdMLP). Reporter gene assays confirm that the region of the AM promoter containing the INR is the target of Myc-mediated repression. Exogenous application of AM peptide to quiescent C3H10T1/2 cultures does not stimulate growth, and constitutive expression of AM mRNA in C3H10T1/2 cells correlates with a reduced potential of the cells to be cotransformed by v-Myc and oncogenic Ras p21. Additional studies showing that AM mRNA is underrepresented in C3H10T1/2 cell lines stably transformed by Ras p21 or adenovirus E1A suggest that AM gene expression is incompatible with deregulated growth in this cell line. We propose a model in which the repression of AM gene expression by Myc is important to the role of this oncoprotein as a potentiator of cellular transformation in C3H10T1/2 and perhaps other cell lines.
An Explanation for Observed Estrogen Receptor Binding to Single-Stranded Estrogen-Responsive Element DNA
Authors: Driscoll Mark D. , Sathya Ganesan , Saidi Layla F. , DeMott Michael S. , Hilf Russell , Bambara Robert A.
Published: January 2014

Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.
Activator Protein-2 Mediates Transcriptional Activation of the CYP11A1 Gene by Interaction with Sp1 Rather than Binding to DNA
Authors: Pena Pilar , Reutens Anne T. , Albanese Chris , D’Amico Mark , Watanabe Genichi , Donner Amy , Shu I-Wei , Williams Trevor , Pestell Richard G.
Published: January 2014

The ovine P450 side chain cleavage (CYP11A1) enzyme gene, which catalyzes the initial enzymatic step in steroid hormone biosynthesis is transcriptionally regulated in cultured steroidogenic human trophoblastic JEG-3 cells. The ovine CYP11A1 promoter contains two GC-rich footprinted regions referred to as ovine footprints 5 (OF5) and OF3, which are well conserved among the CYP11A1 promoters of different species. These GC-rich sequences resemble activator protein-2 (AP-2)/Sp1 binding sites and were previously implicated in basal and cAMP-regulated activity of the bovine and ovine CYP11A1 promoters. In the current studies, AP-2 induced the ovine CYP11A1 promoter 4.5-fold in JEG-3 cells with full induction requiring the previously defined cAMP-responsive elements. Point mutation of OF3 abolished induction by AP-2, and OF3 was sufficient for induction by AP-2 when linked to a heterologous promoter. AP-2 induction of the CYP11A1 promoter required the basic region (N165-N278) and the carboxy terminus of AP-2 (N413-N437). In the course of investigating the mechanisms by which OF5 and OF3 regulated CYP11A1 transcription, we found that OF5 and OF3 bound Sp1 and Sp3 in JEG-3 cells. AP-2 did not bind OF5 or OF3 directly but rather formed a multiprotein complex with Sp1 in JEG-3 cells. AP-2 associated directly with Sp1 in vitro requiring the AP-2 basic region and the Sp1 carboxy terminus. AP-2 induced Sp1/Sp3 activity independently of AP-2 binding to DNA using a GAL4 paradigm. The Sp1 and Sp3 transactivation domains were linked to the DNA-binding domain of GAL4, and their activity was assessed using a luciferase reporter gene containing only the GAL4 DNA-binding sites linked to the minimal TATA site. AP-2 induced Sp1/Sp3-GAL4 activity 3- to 4-fold, requiring both the amino and extreme carboxy terminus of AP-2. We conclude that AP-2 can bind to and stimulate Sp1 activity and induces the ovine CYP11A1 promoter through conserved Sp1/Sp3-binding sites in JEG-3 cells. The induction of Sp1 activity by AP-2 may contribute to the induction of other genes that bind Sp1.
A Synthetic Triterpenoid, 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO), Is a Ligand for the Peroxisome Proliferator-Activated Receptor γ
Authors: Wang Yongping , Porter Weston W. , Suh Nanjoo , Honda Tadashi , Gribble Gordon W. , Leesnitzer Lisa M. , Plunket Kelli D. , Mangelsdorf David J. , Blanchard Steven G. , Willson Timothy M. , Sporn Michael B.
Published: January 2014

A novel synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), previously reported to have potent differentiating, antiproliferative, and antiinflammatory activities, has been identified as a ligand for the peroxisome proliferator-activated receptor γ (PPARγ). CDDO induces adipocytic differentiation in 3T3-L1 cells, although it is not as potent as the full agonist of PPARγ, rosiglitazone. Binding studies of CDDO to PPARγ using a scintillation proximity assay give a Ki between 10−8 to 10−7m. In transactivation assays, CDDO is a partial agonist for PPARγ. The methyl ester of CDDO, CDDO-Me, binds to PPARγ with similar affinity, but is an antagonist. Like other PPARγ ligands, CDDO synergizes with a retinoid X receptor (RXR)-specific ligand to induce 3T3-L1 differentiation, while CDDO-Me is an antagonist in this assay. The partial agonism of CDDO and the antagonism of CDDO-Me reflect the differences in their capacity to recruit or displace cofactors of transcriptional regulation; CDDO and rosiglitazone both release the nuclear receptor corepressor, NCoR, from PPARγ, while CDDO-Me does not. The differences between CDDO and rosiglitazone as either partial or full agonists, respectively, are seen in the weaker ability of CDDO to recruit the coactivator CREB-binding protein, CBP, to PPARγ. Our results establish the triterpenoid CDDO as a member of a new class of PPARγ ligands.
GCN5 and ADA Adaptor Proteins Regulate Triiodothyronine/GRIP1 and SRC-1 Coactivator-Dependent Gene Activation by the Human Thyroid Hormone Receptor
Authors: Anafi Mordecai , Yang Yong-Fan , Barlev Nick A. , Govindan Manjapra V. , Berger Shelley L. , Butt Tauseef R. , Walfish Paul G.
Published: January 2014

We have used yeast genetics and in vitro protein-protein interaction experiments to explore the possibility that GCN5 (general control nonrepressed protein 5) and several other ADA (alteration/deficiency in activation) adaptor proteins of the multimeric SAGA complex can regulate T3/GRIP1 (glucocorticoid receptor interacting protein 1) and SRC-1 (steroid receptor coactivator-1) coactivator-dependent activation of transcription by the human T3 receptor β1 (hTRβ1). Here, we show that in vivo activation of a T3/GRIP1 or SRC-1 coactivator-dependent T3 hormone response element by hTRβ1 is dependent upon the presence of yeast GCN5, ADA2, ADA1, or ADA3 adaptor proteins and that the histone acetyltransferase (HAT) domains and bromodomain (BrD) of yGCN5 must be intact for maximal activation of transcription. We also observed that hTRβ1 can bind directly to yeast or human GCN5 as well as hADA2, and that the hGCN5387−837 sequence could bind directly to either GRIP1 or SRC-1 coactivator. Importantly, the T3-dependent binding of hTRβ1to hGCN5387−837 could be markedly increased by the presence of GRIP1 or SRC1. Mutagenesis of GRIP1 nuclear receptor (NR) Box II and III LXXLL motifs also substantially decreased both in vivo activation of transcription and in vitro T3-dependent binding of hTRβ1 to hGCN5. Taken together, these experiments support a multistep model of transcriptional initiation wherein the binding of T3 to hTRβ1 initiates the recruitment of p160 coactivators and GCN5 to form a trimeric transcriptional complex that activates target genes through interactions with ADA/SAGA adaptor proteins and nucleosomal histones.
β-Cell Differentiation from a Human Pancreatic Cell Line in Vitro and in Vivo
Authors: Dufayet de la Tour Dominique , Halvorsen Tanya , Demeterco Carla , Tyrberg Björn , Itkin-Ansari Pamela , Loy Mary , Yoo Soon-Jib , Hao Ergeng , Bossie Stuart , Levine Fred
Published: January 2014

Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human β-cells, inducibly transformed pancreatic β-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of β-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional β-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human β-cells is a major step toward developing a cell transplantation therapy for diabetes.