Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore.

PubMed

Galigniana, Mario D; Echeverría, Pablo C; Erlejman, Alejandra G; Piwien-Pilipuk, Graciela

2010-01-01

In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR) -domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor "transformation") is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore.

Steroid production and estrogen binding in flowers of Gladiolus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adler, J.H.; Wolfe, G.R.; Janik, J.R.

1987-04-01

The bioconversion of /sup 3/H-cholesterol to steroids was examined in excised tissue from the pistils and bracts of Gladiolus. Ovary-ovule and stigma-style tissues produce a compound with chromatographic properties on reverse phase HPLC similar to 17..beta..-estradiol (E/sub 2/). The stigma-style fraction also produced a compound that chromatographed similarly to progesterone. Bracts and the oxidation controls produced no radiolabeled compounds which were chromatographically similar to E/sub 2/. An endogenous E/sub 2/ binding protein was partially characterized from the ovules. The protein binds E/sub 2/, estriol, and diethylstilbesterol whereas testosterone and progesterone do not bind. The total specific binding capacities in themore » cytosolic and nuclear fractions are 1.6 and 2.2 femtomoles of estradiol per mg of tissue. The dissociation constant is 1.1 x 10/sup -9/ M/sup -1/ for both subcellular fractions. The protein-estradiol complex has a sedimentation coefficient of 4.7 +/- 0.1S. The tissue specific biosynthesis of estrogens and the presence of a steroid binding protein similar to a Type 1 estrogen receptor found in mammals is suggestive of a role for steroids in pistil ontogeny.« less

Determination of association constants between steroid compounds and albumins by partial-filling ACE.

PubMed

Amundsen, Lotta K; Sirén, Heli

2007-10-01

ACE is a popular technique for evaluating association constants between drugs and proteins. However, ACE has not previously been applied to study the association between electrically neutral biomolecules and plasma proteins. We studied the affinity between human and bovine serum albumins (HSA and BSA, respectively) and three neutral endogenous steroid hormones (testosterone, epitestosterone and androstenedione) and two synthetic analogues (methyltestosterone and fluoxymesterone) by applying the partial-filling technique in ACE (PF-ACE). From the endocrinological point of view, the distribution of endogenous steroids among plasma components is of great interest. Strong interactions with albumins suppress the biological activity of steroids. Notable differences in the association constants were observed. In the case of the endogenous steroids, the interactions between testosterone and the albumins were strongest, and those between androstenedione and the albumins were substantially weaker. The association constants, K(b), for testosterone, epitestosterone and androstenedione and HSA at 37 degrees C were 32 100 +/- 3600, 21 600 +/- 1500 and 13 300 +/- 1300 M(-1), respectively, while the corresponding values for the steroids and BSA were 18 800 +/- 1500, 14 000 +/- 400 and 7800 +/- 900 M(-1). Methyltestosterone was bound even more strongly than testosterone, while fluoxymesterone was only weakly bound by the albumins. Finally, the steroids were separated by PF-ACE with HSA and BSA used as resolving components.

Identification of a potential tumor differentiation factor receptor candidate in prostate cancer cells.

PubMed

Sokolowska, Izabela; Woods, Alisa G; Gawinowicz, Mary Ann; Roy, Urmi; Darie, Costel C

2012-07-01

Tumor differentiation factor (TDF) is a pituitary protein that is secreted into the bloodstream and has an endocrine function. TDF and TDF-P1, a 20-residue peptide selected from the ORF of TDF, induce differentiation in human breast and prostate cancer cells, but not in other cells. TDF has no known mechanism of action. In our recent study, we identified heat shock 70 kDa proteins (HSP70s) as TDF receptors (TDF-Rs) in breast cancer cells. Therefore, we sought to investigate whether TDF-R candidates from prostate cancer cells are the same as those identified in breast cancer cells. Here, we used TDF-P1 to purify the potential TDF-R candidates by affinity purification chromatography from DU145 and PC3 steroid-resistant prostate cancer cells, LNCaP steroid-responsive prostate cancer cells, and nonprostate NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. We identified the purified proteins by MS, and validated them by western blotting, immunofluorescence microscopy, immunoaffinity purification chromatography, and structural biology. We identified seven candidate proteins, of which three were from the HSP70 family. These three proteins were validated as potential TDF-R candidates in LNCaP steroid-responsive and in DU145 and PC3 steroid-resistant prostate cancer cells, but not in NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. Our previous study and the current study suggest that GRP78, and perhaps HSP70s, are strong TDF-R candidates, and further suggest that TDF interacts with its receptors exclusively in breast and prostate cells, inducing cell differentiation through a novel, steroid-independent pathway. © 2012 The Authors Journal compilation © 2012 FEBS.

Steroids and endocrine disruptors--History, recent state of art and open questions.

PubMed

Hampl, Richard; Kubátová, Jana; Stárka, Luboslav

2016-01-01

This introductory chapter provides an overview of the levels and sites at which endocrine disruptors (EDs) affect steroid actions. In contrast to the special issue of Journal of Steroid Biochemistry and Molecular Biology published three years ago and devoted to EDs as such, this paper focuses on steroids. We tried to point to more recent findings and opened questions. EDs interfere with steroid biosynthesis and metabolism either as inhibitors of relevant enzymes, or at the level of their expression. Particular attention was paid to enzymes metabolizing steroid hormones to biologically active products in target cells, such as aromatase, 5α-reductase and 3β-, 11β- and 17β-hydroxysteroid dehydrogenases. An important target for EDs is also steroid acute regulatory protein (StAR), responsible for steroid precursor trafficking to mitochondria. EDs influence receptor-mediated steroid actions at both genomic and non-genomic levels. The remarkable differences in response to various steroid-receptor ligands led to a more detailed investigation of events following steroid/disruptor binding to the receptors and to the mapping of the signaling cascades and nuclear factors involved. A virtual screening of a large array of EDs with steroid receptors, known as in silico methods (≡computer simulation), is another promising approach for studying quantitative structure activity relationships and docking. New data may be expected on the effect of EDs on steroid hormone binding to selective plasma transport proteins, namely transcortin and sex hormone-binding globulin. Little information is available so far on the effects of EDs on the major hypothalamo-pituitary-adrenal/gonadal axes, of which the kisspeptin/GPR54 system is of particular importance. Kisspeptins act as stimulators for hormone-induced gonadotropin secretion and their expression is regulated by sex steroids via a feed-back mechanism. Kisspeptin is now believed to be one of the key factors triggering puberty in mammals, and various EDs affect its expression and function. Finally, advances in analytics of EDs, especially those persisting in the environment, in various body fluids (plasma, urine, seminal fluid, and follicular fluid) are mentioned. Surprisingly, relatively scarce information is available on the simultaneous determination of EDs and steroids in the same biological material. This article is part of a Special Issue entitled 'Endocrine disruptors & steroids'. Copyright © 2014 Elsevier Ltd. All rights reserved.

Anabolic steroid abuse and tooth size-arch dimensions in the rat.

PubMed

Barrett, R L; Harris, E F; Tolley, E A; Nutting, D F

1993-01-01

Anabolic steroids are misused by adolescents and adults to increase muscle mass and improve appearance and athletic performance. Since anabolics strongly enhance protein synthesis, it was speculated that alterations in tooth size and arch length could occur. This study quantified the effects of the anabolic steroid nandrolone phenpropionate on these parameters in a rat model. The steroid significantly increased mandibular arch length. No difference in mesiodistal dimensions of the molars occurred. In consequence, the increased arch dimensions combined with unaltered tooth size may result in dental spacing and/or other malocclusions.

Sex steroid-induced changes in circulating monocyte chemoattractant protein-1 levels may contribute to metabolic dysfunction in obese men.

PubMed

Ruige, Johannes B; Bekaert, Marlies; Lapauw, Bruno; Fiers, Tom; Lehr, Stefan; Hartwig, Sonja; Herzfeld de Wiza, Daniella; Schiller, Martina; Passlack, Waltraud; Van Nieuwenhove, Yves; Pattyn, Piet; Cuvelier, Claude; Taes, Youri E; Sell, Henrike; Eckel, Juergen; Kaufman, Jean-Marc; Ouwens, D Margriet

2012-07-01

Low testosterone accompanied by elevated estradiol associates with the development of metabolic dysfunction in men. The aim of the study was to explore the hypothesis that alterations in sex steroid levels induce metabolic dysfunction through adipokines. Circulating levels of sex steroids and 28 adipokines were determined in a cross-sectional study of morbidly obese men and aged-matched controls, as well as in a randomized clinical trial with healthy young men in which obesity-related alterations in sex steroid levels were mimicked by treatment with an aromatase inhibitor plus estradiol patches. Morbidly obese men had lower testosterone levels than normal-weight controls. Estradiol levels were increased in morbidly obese men (without DM2) as compared to normal-weight controls. Circulating levels of multiple proinflammatory cytokines, including IL-1Ra, IL-5, IL-6, IL-10, leptin, monocyte chemoattractant protein 1 (MCP1), and macrophage inflammatory protein 1α, positively associated with estradiol and negatively with testosterone. The associations with estradiol, but not with testosterone, remained significant after adjusting for adipocyte cell size. In a separate clinical trial, the direct adverse effects of lowering testosterone and raising estradiol on MCP1 were substantiated in vivo. Initial alterations in sex steroid levels may contribute to metabolic dysfunction through adverse effects on adipokine levels in obese men. The direct adverse effects on MCP1, a chemokine highly linked to the development of metabolic dysfunction, were substantiated in a trial mimicking obesity-related alterations of sex steroid levels in healthy young males.

Regucalcin Expression in Bovine Tissues and Its Regulation by Sex Steroid Hormones in Accessory Sex Glands

PubMed Central

Starvaggi Cucuzza, Laura; Divari, Sara; Mulasso, Chiara; Biolatti, Bartolomeo; Cannizzo, Francesca T.

2014-01-01

Regucalcin (RGN) is a mammalian Ca2+-binding protein that plays an important role in intracellular Ca2+ homeostasis. Recently, RGN has been identified as a target gene for sex steroid hormones in the prostate glands and testis of rats and humans, but no studies have focused on RGN expression in bovine tissues. Thus, in the present study, we examined RGN mRNA and protein expression in the different tissues and organs of veal calves and beef cattle. Moreover, we investigated whether RGN expression is controlled through sex steroid hormones in bovine target tissues, namely the bulbo-urethral and prostate glands and the testis. Sex steroid hormones are still illegally used in bovine husbandry to increase muscle mass. The screening of the regulation and function of anabolic sex steroids via modified gene expression levels in various tissues represents a new approach for the detection of illicit drug treatments. Herein, we used quantitative PCR, western blot and immunohistochemistry analyses to demonstrate RGN mRNA and protein expression in bovine tissues. In addition, estrogen administration down-regulated RGN gene expression in the accessory sex glands of veal calves and beef cattle, while androgen treatment reduced RGN gene expression only in the testis. The confirmation of the regulation of RGN gene expression through sex steroid hormones might facilitate the potential detection of hormone abuse in bovine husbandry. Particularly, the specific response in the testis suggests that this tissue is ideal for the detection of illicit androgen administration in veal calves and beef cattle. PMID:25415588

Isoform composition and stoichiometry of the approx. 90-kDa heat shock protein associated with glucocorticoid receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendel, D.B.; Orti, E.

1988-05-15

The authors observed that the approx. 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approx. 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approx. 90-kDa heat shock protein. The observation that TSTA and the approx. 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested thatmore » the doublet observed is also due to the existence of two isoforms. They have therefore conducted this study to determine whether TSTA and the approx. 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approx. 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. They used the BuGr1 and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approx. 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with (/sup 35/S)methionine to metabolically label proteins to steady state. The long-term metabolic labeling approach has also enabled them to directly determine that the purified non-activated glucocorticoid receptor contains a single steroid-binding protein and two approx. 90-kDa non-steroid-binding subunits. The consistency with which a approx. 1:2 stoichiometric ratio of steroid binding to approx. 90-kDa protein is observed supports the view that the approx. 90-kDa heat shock protein is a true component of nonactivated glucocorticoid-receptor complexes.« less

What goes on behind closed doors: physiological vs. pharmacological steroid hormone actions

PubMed Central

Simons, S. Stoney

2009-01-01

Summary Steroid hormone-activated receptor proteins are among the best understood class of factors for altering gene transcription in cells. Steroid receptors are of major importance in maintaining normal human physiology by responding to circulating concentrations of steroid in the nM range. Nonetheless, most studies of steroid receptor action have been conducted using the supra-physiological conditions of saturating concentrations (≥100 nM) of potent synthetic steroid agonists. Here we summarize the recent developments arising from experiments using two clinically relevant conditions: subsaturating concentrations of agonist (to mimic the circulating concentrations in mammals) and saturating concentrations of antagonists (which are employed in endocrine therapies to block the actions of endogenous steroids). These studies have revealed new facets of steroid hormone action that could not be uncovered by conventional experiments with saturating concentrations of agonist steroids, such as a plethora of factors/conditions for the differential control of gene expression by physiological levels of steroid, a rational approach for examining the gene-specific variations in partial agonist activity of antisteroids, and a dissociation of steroid potency and efficacy that implies the existence of separate, and possibly novel, mechanistic steps and cofactors. PMID:18623071

The Unique Dopamine/Ecdysteroid Receptor Modulates Ethanol-Induced Sedation in Drosophila.

PubMed

Petruccelli, Emily; Li, Qi; Rao, Yi; Kitamoto, Toshihiro

2016-04-20

Steroids profoundly influence behavioral responses to alcohol by activating canonical nuclear hormone receptors and exerting allosteric effects on ion channels. Accumulating evidence has demonstrated that steroids can also trigger biological effects by directly binding G-protein-coupled receptors (GPCRs), yet physiological roles of such unconventional steroid signaling in controlling alcohol-induced behaviors remain unclear. The dopamine/ecdysteroid receptor (DopEcR) is a GPCR that mediates nongenomic actions of ecdysteroids, the major steroid hormones in insects. Here, we report that Drosophila DopEcR plays a critical role in ethanol-induced sedation.DopEcR mutants took longer than control flies to become sedated during exposure to ethanol, despite having normal ethanol absorption or metabolism. RNAi-mediated knockdown of DopEcR expression revealed that this receptor is necessary after eclosion, and is required in particular neuronal subsets, including cholinergic and peptidergic neurons, to mediate this behavior. Additionally, flies ubiquitously overexpressing DopEcR cDNA had a tendency to become sedated quickly upon ethanol exposure. These results indicate that neuronal subset-specific expression of DopEcR in adults is required for normal sedation upon exposure to ethanol. We also obtained evidence indicating that DopEcR may promote ethanol sedation by suppressing epidermal growth factor receptor/extracellular signal-regulated kinase signaling. Last, genetic and pharmacological analyses suggested that in adult flies ecdysone may serve as an inverse agonist of DopEcR and suppress the sedation-promoting activity of DopEcR in the context of ethanol exposure. Our findings provide the first evidence for the involvement of nongenomic G-protein-coupled steroid receptors in the response to alcohol, and shed new light on the potential roles of steroids in alcohol-use disorders. Alcohol abuse is an alarming personal and societal burden. The improvement of prevention and treatment strategies for alcohol-use disorders requires a better understanding of their biological basis. Steroid hormones profoundly affect alcohol-induced behaviors, but the contribution of their unconventional, nongenomic actions during these responses has not yet been elucidated. We found that Drosophila DopEcR, a unique G-protein-coupled receptor (GPCR) with dual specificity for dopamine and steroids, mediates noncanonical steroid actions to promote ethanol-induced sedation. Because steroid signaling and the behavioral response to alcohol are evolutionarily well conserved, our findings suggest that analogous mammalian receptors likely play important roles in alcohol-use disorders. Our work provides a foundation for further characterizing the function and mechanisms of action of nonclassical steroid GPCR signaling. Copyright © 2016 the authors 0270-6474/16/364647-11$15.00/0.

The Unique Dopamine/Ecdysteroid Receptor Modulates Ethanol-Induced Sedation in Drosophila

PubMed Central

Petruccelli, Emily; Li, Qi; Rao, Yi

2016-01-01

Steroids profoundly influence behavioral responses to alcohol by activating canonical nuclear hormone receptors and exerting allosteric effects on ion channels. Accumulating evidence has demonstrated that steroids can also trigger biological effects by directly binding G-protein-coupled receptors (GPCRs), yet physiological roles of such unconventional steroid signaling in controlling alcohol-induced behaviors remain unclear. The dopamine/ecdysteroid receptor (DopEcR) is a GPCR that mediates nongenomic actions of ecdysteroids, the major steroid hormones in insects. Here, we report that Drosophila DopEcR plays a critical role in ethanol-induced sedation. DopEcR mutants took longer than control flies to become sedated during exposure to ethanol, despite having normal ethanol absorption or metabolism. RNAi-mediated knockdown of DopEcR expression revealed that this receptor is necessary after eclosion, and is required in particular neuronal subsets, including cholinergic and peptidergic neurons, to mediate this behavior. Additionally, flies ubiquitously overexpressing DopEcR cDNA had a tendency to become sedated quickly upon ethanol exposure. These results indicate that neuronal subset-specific expression of DopEcR in adults is required for normal sedation upon exposure to ethanol. We also obtained evidence indicating that DopEcR may promote ethanol sedation by suppressing epidermal growth factor receptor/extracellular signal-regulated kinase signaling. Last, genetic and pharmacological analyses suggested that in adult flies ecdysone may serve as an inverse agonist of DopEcR and suppress the sedation-promoting activity of DopEcR in the context of ethanol exposure. Our findings provide the first evidence for the involvement of nongenomic G-protein-coupled steroid receptors in the response to alcohol, and shed new light on the potential roles of steroids in alcohol-use disorders. SIGNIFICANCE STATEMENT Alcohol abuse is an alarming personal and societal burden. The improvement of prevention and treatment strategies for alcohol-use disorders requires a better understanding of their biological basis. Steroid hormones profoundly affect alcohol-induced behaviors, but the contribution of their unconventional, nongenomic actions during these responses has not yet been elucidated. We found that Drosophila DopEcR, a unique G-protein-coupled receptor (GPCR) with dual specificity for dopamine and steroids, mediates noncanonical steroid actions to promote ethanol-induced sedation. Because steroid signaling and the behavioral response to alcohol are evolutionarily well conserved, our findings suggest that analogous mammalian receptors likely play important roles in alcohol-use disorders. Our work provides a foundation for further characterizing the function and mechanisms of action of nonclassical steroid GPCR signaling. PMID:27098705

P-Glycoprotein Activity in Steroid-Responsive vs. Steroid-Resistant Nephrotic Syndrome.

PubMed

Badr, Hassan S; El-Hawy, Mahmoud A; Helwa, Mohammed A

2016-11-01

To explore the expression of P-glycoprotein (P-gp) in the peripheral blood nucleated cells (PBNCs) of children with nephrotic syndrome in relation to their clinical response to glucocorticoid treatment. Thirty-six children with nephrotic syndrome (20 cases of steroid-responsive and 16 cases of steroid-resistant) were examined. All the participants were subjected to complete history taking, thorough clinical examination, laboratory investigations (24-h urinary protein, serum albumin, complete blood count with differential white blood cell count, serum cholesterol, serum urea, serum creatinine) and functional assay of P-gp using FACS Calibur flowcytometry. P-gp assay was done in both groups during remission. P-gp activity was significantly higher in steroid-resistant than steroid-sensitive cases. P-gp can be used as a predictor of outcome, as a part of laboratory evaluation of the cases before starting steroid therapy, so as to determine whether to use alternative line of therapy or use one of the P-gp inhibitors with steroid therapy.

Sex-hormone-binding globulin.

PubMed

Anderson, D C

1974-01-01

A review was made to understand how plasma binding protein might influence sex-hormone action in target tissues. Steroids are predominately bound to plasma proteins and only unbound steroids enter the cells. Sex-hormone-binding globulin (SHBG) binds to both the main circulating steroid T and E2 but changes in SHBG concentrations exert significant results. Increased SHBG levels increase estrogen production and decreases T activity; whereas, increased androgens increase T action and inhibit SHBG production. These disturbances in hormone maintenance may lead to abnormal adult sex differentiation such as hirsutism and forms of hynaecomastia. By developing SHBG concentration measurement methods-responses of hirsutism to glucocorticoid or estrogem may be assessed. In addition, the effect of thyroid hormones on SHBG may also have therapeutic implications in endocrine disease.

Exposure to media predicts use of dietary supplements and anabolic-androgenic steroids among Flemish adolescent boys.

PubMed

Frison, Eline; Vandenbosch, Laura; Eggermont, Steven

2013-10-01

This study examined whether different types of media affect the use of dietary proteins and amino acid supplements, and intent to use anabolic-androgenic steroids. A random sample of 618 boys aged 11-18 years from eight schools in the Flemish part of Belgium completed standardized questionnaires as part of the Media and Adolescent Health Study. The survey measured exposure to sports media, appearance-focused media, fitness media, use of dietary supplements, and intent to use anabolic-androgenic steroids. Data were analyzed using logistic regressions and are presented as adjusted odds ratios (OR) and 95 % confidence intervals (CI); 8.6 % indicated to have used dietary proteins, 3.9 % indicated to have used amino acid supplements, and 11.8 % would consider using anabolic-androgenic steroids. After adjusting for fitness activity, exposure to fitness media was associated with the use of dietary proteins (OR = 7.24, CI = 2.25-23.28) and amino acid supplements (5.16, 1.21-21.92; 44.30, 8.25-238). Intent to use anabolic-androgenic steroids was associated with exposure to fitness media (2.38, 1.08-5.26; 8.07, 2.55-25.53) and appearance-focused media (6.02, 1.40-25.82; 8.94, 1.78-44.98). Sports media did not correlate with the use of dietary supplements and intent to use anabolic-androgenic steroids. Specific types of media are strong predictors of the use of supplements in adolescent boys. This provides an opportunity for intervention and prevention through the selection of fitness media as a communication channel. Health practitioners should also be aware that the contemporary body culture exerts pressure not only on girls but also on boys.

Occurrence, source analysis and risk assessment of androgens, glucocorticoids and progestagens in the Hailing Bay region, South China Sea.

PubMed

Liu, Shan; Chen, Hui; Zhou, Guang-Jie; Liu, Shuang-Shuang; Yue, Wei-Zhong; Yu, Shen; Sun, Kai-Feng; Cheng, Hefa; Ying, Guang-Guo; Xu, Xiang-Rong

2015-12-01

The occurrence and spatial distribution of 40 steroids in the environmental matrices of the Hailing Bay region, South China Sea, were investigated by rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS). Seventeen, 14 and 11 of 40 steroids were detected with the concentrations ranging from 0.04 (testosterone) to 40.00 ng/L (prednisolone), 1.33 (4-hydroxy-androst-4-ene-17-dione) to 1855 ng/L (androsta-1,4-diene-3,17-dione) and <0.19 (androsta-1,4-diene-3,17-dione) to 2.37 ng/g (progesterone) in the seawater, the municipal sewage discharged effluent and the sediment samples, respectively. The concentrations and risk quotients (RQs) of the steroids detected in the water samples decreased in the order of municipal sewage discharge site>wharves~aquaculture zones~tourism areas>offshore areas. The distribution of steroids in the marine environment was significantly correlated with the levels of chemical oxygen demand (COD) and ammonium nitrogen (NH4-N). Source analysis indicated that untreated municipal sewage was the main source of steroids in the marine environment. Furthermore, progesterone was found to be a reliable chemical indicator to surrogate different steroids in both the water and sediment phases based on the correlation analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Steroid-Responsive Recurrent Encephalopathy Associated with Subacute Thyroiditis

PubMed Central

Chung, Yun Jae; Ahn, Jihyun; Ha, Sam-Yeol; Youn, Young Chul

2008-01-01

Background Steroid-responsive encephalopathy associated with subacute thyroiditis has, to our knowledge, not been reported previously. Case Report A 49-year-old woman was found collapsed and brought to our institution with decreased mentality, dysarthria, and gait disturbance. Brain magnetic resonance imaging and angiography were normal but blood tests revealed thyroid-autoantibody-negative thyrotoxicosis. Results of a 99mtechnetium-pertechnetate scan were compatible with the thyrotoxic phase of subacute thyroiditis. 14-3-3 proteins were detected in cerebrospinal fluid. Her mental status began to improve from the day following steroid administration. Recurrent encephalopathy was found 2 months after the initial admission, which was also effectively treated with steroid. Conclusions We speculate that steroid-responsive recurrent encephalopathy associated with subacute thyroiditis is a subtype of Hashimoto's encephalopathy, and consider that steroid treatment should not be delayed in suspected patients. PMID:19513293

Evaluation of anabolic steroid induced renal damage with sonography in bodybuilders.

PubMed

Kantarci, Umut H; Punduk, Zekine; Senarslan, Omer; Dirik, Alper

2017-11-17

The aim of this study was to investigate the effect of anabolic steroids on kidneys in bodybuilders. Twenty two bodybuilders were included in the study. Participants were divided into three groups according to the scheme of steroid usage: Group 1 (n=8, intramuscular 500 mg testosterone enanthate, intramuscular 400 mg nandrolone decanoate and oral 40 mg methandrostenolone for 12 weeks), Group 2 (n=7, intramuscular 500 mg testosterone enanthate, intramuscular 300 mg nandrolone decanoate and intramuscular 300 mg boldenone undecylenate for 16 weeks) and Group 3 (n=7, no steroid intake). Blood urea nitrogen (BUN), creatinine (Cr), urine microalbumin and electrolyte levels were measured. Renal volume, cortical thickness and echogenicity were obtained in ultrasonographic scans. Renal volume, cortical thickness, echogenicity and protein intake value were significantly higher in group 2 than group 1 and 3. Plasma levels of BUN and Cr in group 2 were significantly higher than other groups (p ˂ 0.001). Urine microalbumin and electrolyte levels were normal in all groups. The results of this study indicate that high protein intake, steroid usage, particularly the schemes, including boldenone undecylenate increases cortical echogenicity, thickness of renal parenchyma and renal volume in bodybuilders.

Binding domain-driven intracellular trafficking of sterols for synthesis of steroid hormones, bile acids and oxysterols.

PubMed

Midzak, Andrew; Papadopoulos, Vassilios

2014-09-01

Steroid hormones, bioactive oxysterols and bile acids are all derived from the biological metabolism of lipid cholesterol. The enzymatic pathways generating these compounds have been an area of intense research for almost a century, as cholesterol and its metabolites have substantial impacts on human health. Owing to its high degree of hydrophobicity and the chemical properties that it confers to biological membranes, the distribution of cholesterol in cells is tightly controlled, with subcellular organelles exhibiting highly divergent levels of cholesterol. The manners in which cells maintain such sterol distributions are of great interest in the study of steroid and bile acid synthesis, as limiting cholesterol substrate to the enzymatic pathways is the principal mechanism by which production of steroids and bile acids is regulated. The mechanisms by which cholesterol moves within cells, however, remain poorly understood. In this review, we examine the subcellular machinery involved in cholesterol metabolism to steroid hormones and bile acid, relating it to both lipid- and protein-based mechanisms facilitating intracellular and intraorganellar cholesterol movement and delivery to these pathways. In particular, we examine evidence for the involvement of specific protein domains involved in cholesterol binding, which impact cholesterol movement and metabolism in steroidogenesis and bile acid synthesis. A better understanding of the physical mechanisms by which these protein- and lipid-based systems function is of fundamental importance to understanding physiological homeostasis and its perturbation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum.

PubMed

Poli, Anna; Di Pietro, Antonio; Zigon, Dusan; Lenasi, Helena

2009-02-01

Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15alpha-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein beta subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Deltafgb1 strain, suggesting a role for the G-protein beta subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Deltafgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.

Keto-supplemented Low Protein Diet: A Valid Therapeutic Approach for Patients with Steroid-resistant Proteinuria during Early-stage Chronic Kidney Disease.

PubMed

Zhang, J; Xie, H; Fang, M; Wang, K; Chen, J; Sun, W; Yang, L; Lin, H

2016-04-01

Low protein diets supplemented with keto acid (sLPD) are recommended for patients with stage 3-5 chronic kidney disease (CKD). This study assessed whether sLPD is beneficial for patients with steroid-resistant proteinuria during early-stage CKD. A 1-year randomized controlled trial was conducted from 2010 to 2012. In this study, 108 proteinuric patients who were steroid-resistant were assigned to a sLPD group (0.6 g/kg/d with 0.09 g/kg/d keto acids) or a normal protein diet group (NPD, 1.0 g/kg/d). Estimated dietary protein intake, urinary protein excretion, remission rate, renal function, nutritional status, and blood pressure were measured. Baseline characteristics were comparable between the sLPD group (47 patients) and the NPD group (49 patients). Urinary protein excretion significantly decreased in sLPD compared to NPD in months 6, 9, and 12 (P<0.05). Proteinuria reduction was higher in sLPD than in NPD (P<0.001) at the end of the study. Complete remission and partial remission rates were higher in sLPD than in NPD. Serum albumin and pre-albumin levels were higher in sLPD than in NPD in months 9 and 12 (P<0.05). Serum total cholesterol and triglyceride levels declined more significantly in sLPD than in NPD (P<0.01) at the end of the study. There were no differences in nutritional status, renal function, hemoglobin, or blood pressure between the two groups. sLPD is both nutritionally safe and beneficial, providing nephroprotective effects for early-stage CKD patients with steroid-resistant proteinuria.

High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells

PubMed Central

Boonyaratanakornkit, Viroj; Melvin, Vida; Prendergast, Paul; Altmann, Magda; Ronfani, Lorenza; Bianchi, Marco E.; Taraseviciene, Laima; Nordeen, Steven K.; Allegretto, Elizabeth A.; Edwards, Dean P.

1998-01-01

We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by several nonsteroid nuclear receptors, including retinoid acid receptor (RAR), retinoic X receptor (RXR), and vitamin D receptor (VDR). As highly purified recombinant full-length proteins, all steroid receptors tested exhibited weak binding affinity for their optimal palindromic hormone response elements (HREs), and the addition of purified HMG-1 or -2 substantially increased their affinity for HREs. Purified RAR, RXR, and VDR also exhibited little to no detectable binding to their cognate direct repeat HREs but, in contrast to results with steroid receptors, the addition of HMG-1 or HMG-2 had no stimulatory effect. Instead, the addition of purified RXR enhanced RAR and VDR DNA binding through a heterodimerization mechanism and HMG-1 or HMG-2 had no further effect on DNA binding by RXR-RAR or RXR-VDR heterodimers. HMG-1 and HMG-2 (HMG-1/-2) themselves do not bind to progesterone response elements, but in the presence of PR they were detected as part of an HMG-PR-DNA ternary complex. HMG-1/-2 can also interact transiently in vitro with PR in the absence of DNA; however, no direct protein interaction was detected with VDR. These results, taken together with the fact that PR can bend its target DNA and that HMG-1/-2 are non-sequence-specific DNA binding proteins that recognize DNA structure, suggest that HMG-1/-2 are recruited to the PR-DNA complex by the combined effect of transient protein interaction and DNA bending. In transient-transfection assays, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in mammalian cells by as much as 7- to 10-fold without altering the basal promoter activity of target reporter genes. This increase in PR-mediated gene activation by coexpression of HMG-1/-2 was observed in different cell types and with different target promoters, suggesting a generality to the functional interaction between HMG-1/-2 and PR in vivo. Cotransfection of HMG-1 also increased reporter gene activation mediated by other steroid receptors, including glucocorticoid and androgen receptors, but it had a minimal influence on VDR-dependent transcription in vivo. These results support the conclusion that HMG-1/-2 are coregulatory proteins that increase the DNA binding and transcriptional activity of the steroid hormone class of receptors but that do not functionally interact with certain nonsteroid classes of nuclear receptors. PMID:9671457

Epigenetic regulation of the expression of genes involved in steroid hormone biosynthesis and action

PubMed Central

Martinez-Arguelles, Daniel B.; Papadopoulos, Vassilios

2010-01-01

Steroid hormones participate in organ development, reproduction, body homeostasis, and stress responses. The steroid machinery is expressed in a development- and tissue-specific manner, with the expression of these factors being tightly regulated by an array of transcription factors (TFs). Epigenetics provides an additional layer of gene regulation through DNA methylation and histone tail modifications. Evidence of epigenetic regulation of key steroidogenic enzymes is increasing, though this does not seem to be a predominant regulatory pathway. Steroid hormones exert their action in target tissues through steroid nuclear receptors belonging to the NR3A and NR3C families. Nuclear receptor expression levels and post-translational modifications regulate their function and dictate their sensitivity to steroid ligands. Nuclear receptors and TFs are more likely to be epigenetically regulated than proteins involved in steroidogenesis and have secondary impact on the expression of these steroidogenic enzymes. Here we review evidence for epigenetic regulation of enzymes, transcription factors, and nuclear receptors related to steroid biogenesis and action. PMID:20156469

Variable steroid receptor responses: Intrinsically disordered AF1 is the key

PubMed Central

Simons, S. Stoney; Kumar, Raj

2013-01-01

Steroid hormones, acting through their cognate receptor proteins, see widespread clinical applications due to their ability to alter the induction or repression of numerous genes. However, steroid usage is limited by the current inability to control off-target, or non-specific, side-effects. Recent results from three separate areas of research with glucocorticoid and other steroid receptors (cofactor-induced changes in receptor structure, the ability of ligands to alter remote regions of receptor structure, and how cofactor concentration affects both ligand potency and efficacy) indicate that a key element of receptor activity is the intrinsically disordered amino-terminal domain. These results are combined to construct a novel framework within which to logically pursue various approaches that could afford increased selectivity in steroid-based therapies. PMID:23792173

The ESC/E(Z) complex, an effector of response to ovarian steroids, manifests an intrinsic difference in cells from women with Premenstrual Dysphoric Disorder

PubMed Central

Dubey, Neelima; Hoffman, Jessica F.; Schuebel, Kornel; Yuan, Qiaoping; Martinez, Pedro E.; Nieman, Lynnette K.; Rubinow, David R.; Schmidt, Peter J.; Goldman, David

2016-01-01

Clinical evidence suggests that mood and behavioral symptoms in Premenstrual Dysphoric Disorder (PMDD), a common, recently recognized, psychiatric condition among women, reflect abnormal responsivity to ovarian steroids. This differential sensitivity could be due to an unrecognized aspect of hormonal signaling or a difference in cellular response. In this study, lymphoblastoid cell line cultures (LCLs) from women with PMDD and asymptomatic Controls were compared via whole transcriptome sequencing (RNA-seq) during untreated (ovarian steroid-free) conditions and following hormone treatment. The women with PMDD manifested ovarian steroid-triggered behavioral sensitivity during a hormone suppression and add-back clinical trial, and Controls did not, leading us to hypothesize that women with PMDD might differ in their cellular response to ovarian steroids. In untreated LCLs, our results overall suggest a divergence between mRNA (e.g., gene transcription) and protein (e.g., RNA translation in proteins) for the same genes. Pathway analysis of the LCL transcriptome revealed, among others, over-expression of ESC/E(Z) complex genes (an ovarian steroid-regulated gene silencing complex) in untreated LCLs from women with PMDD, with more than half of these genes over-expressed as compared to Controls, and with significant effects for MTF2, PHF19, and SIRT1 (p<0.05). RNA and protein expression of the 13 ESC/E(Z) complex genes were individually quantitated. This pattern of increased ESC/E(Z) mRNA expression was confirmed in a larger cohort by qRT-PCR. In contrast, protein expression of ESC/E(Z) genes was decreased in untreated PMDD LCLs with MTF2, PHF19, and SIRT1 all significantly decreased (p<0.05). Finally, mRNA expression of several ESC/E(Z) complex genes were increased by progesterone in Controls only, and decreased by estradiol in PMDD LCLs. These findings demonstrate that LCLs from women with PMDD manifest a cellular difference in ESC/E(Z) complex function both in the untreated condition and in response to ovarian hormones. Dysregulation of ESC/E(Z) complex function could contribute to PMDD. PMID:28044059

Steroid toxicity and detoxification in ascomycetous fungi.

PubMed

Cvelbar, Damjana; Zist, Vanja; Kobal, Katja; Zigon, Dušan; Zakelj-Mavrič, Marija

2013-02-25

In the last couple of decades fungal infections have become a significant clinical problem. A major interest into fungal steroid action has been provoked since research has proven that steroid hormones are toxic to fungi and affect the host/fungus relationship. Steroid hormones were found to differ in their antifungal activity in ascomycetous fungi Hortaea werneckii, Saccharomyces cerevisiae and Aspergillus oryzae. Dehydroepiandrosterone was shown to be the strongest inhibitor of growth in all three varieties of fungi followed by androstenedione and testosterone. For their protection, fungi use several mechanisms to lower the toxic effects of steroids. The efficiency of biotransformation in detoxification depended on the microorganism and steroid substrate used. Biotransformation was a relatively slow process as it also depended on the growth phase of the fungus. In addition to biotransformation, steroid extrusion out of the cells contributed to the lowering of the active intracellular steroid concentration. Plasma membrane Pdr5 transporter was found to be the most effective, followed by Snq2 transporter and vacuolar transporters Ybt1 and Ycf1. Proteins Aus1 and Dan1 were not found to be involved in steroid import. The research of possible targets of steroid hormone action in fungi suggests that steroid hormones inhibit ergosterol biosynthesis in S. cerevisiae and H. werneckii. Results of this inhibition caused changes in the sterol content of the cellular membrane. The presence of steroid hormones most probably causes the degradation of the Tat2 permease and impairment of tryptophan import. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Modifications of Western-type diet regarding protein, fat and sucrose levels as modulators of steroid metabolism and activity in liver.

PubMed

Krawczyńska, Agata; Herman, Andrzej P; Antushevich, Hanna; Bochenek, Joanna; Dziendzikowska, Katarzyna; Gajewska, Alina; Gromadzka-Ostrowska, Joanna

2017-01-01

The aim of the study was to evaluate whether the modification of the Western-type diet (high-fat, high-sucrose diet rich in saturated fatty acids) considering macronutrients content would influence hepatic metabolism and activity of steroids. For 3 weeks Wistar rat were fed the Western-type diet (21% fat, 35% sucrose, 19% protein, lard) and its modifications regarding dietary protein (10 and 19%), fat (5 and 21%) and sucrose (0 and 35%) levels. The steroid 5α-reductase type 1 (Srd5a1) and androgen receptor (Ar) gene expression as well as testosterone (T) conversion towards 5α-reduced derivatives in liver were positively correlated with body weight gain. The Western-type diets with decreased protein content regardless of the sucrose level exerted the most negative effect on the antioxidant system decreasing catalase (Cat), sodium dismutase (Sod1) and glutathione peroxidase (Gpx1) gene expression as well as Cat and Gpx activity and total antioxidant status, simultaneously intensifying lipid peroxidation. The impaired antioxidant system was accompanied by decreased level of hepatic T metabolism towards estrogens: 17β-estradiol (E2) and estriol, and increased estrogen receptor type 1 (Esr1) gene expression. Liver Esr1 mRNA level was differently correlated with T (positively) and E2 (negatively) plasma levels. Whereas the fat reduction in Western-type diet restored the plasma proportion between T and E2. In conclusion it could be stated that Western-type diet modification relating to protein, sucrose and fat content can influence hepatic steroid metabolism and activity; however the estrogens and androgens metabolism in liver would be connected with impairment of liver function or catabolic activity, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mesalamine Intolerance in Three Children with Crohn's Disease.

PubMed

Tuna Kirsaclioglu, Ceyda; Kuloglu, Zarife; Ustundag, Gonca; Kansu, Aydan; İnce, Erdal; Ensari, Arzu; Girgin, Nurten

2016-01-01

To present the mesalamine-induced acute exacerbation of symptoms and inflammatory markers in children with Crohn's disease (CD). Three children who presented with CD had acute exacerbation of colitis symptoms or elevated inflammatory markers when mesalamine was added to treatment while tapering/ceasing steroid treatment. While on steroid treatment, the patients maintained clinical and laboratory remission, but with the initiation of mesalamine treatment, they had abdominal pain and bloody mucoid diarrhoea and/or elevation of white blood cell count, C-reactive protein level and erythrocyte sedimentation rate. Bacterial pathogens were excluded from the urine, throat and blood cultures, parasites with stool examination, viral pathogens with serology. Within 3-7 days after the mesalamine treatment had been stopped, the patients showed improvement of colitis symptoms and normalisation of white blood cell count, C-reactive protein level and erythrocyte sedimentation rate. In this study mesalamine mimicked CD relapse in children with CD while tapering or after stopping steroid treatment. Awareness of this side effect of mesalamine could prevent a misdiagnosis of steroid dependency. © 2015 S. Karger AG, Basel.

Mesalamine Intolerance in Three Children with Crohn's Disease

PubMed Central

Tuna Kirsaclioglu, Ceyda; Kuloglu, Zarife; Ustundag, Gonca; Kansu, Aydan; İnce, Erdal; Ensari, Arzu; Girgin, Nurten

2016-01-01

Objective To present the mesalamine-induced acute exacerbation of symptoms and inflammatory markers in children with Crohn's disease (CD). Clinical Presentation and Intervention Three children who presented with CD had acute exacerbation of colitis symptoms or elevated inflammatory markers when mesalamine was added to treatment while tapering/ceasing steroid treatment. While on steroid treatment, the patients maintained clinical and laboratory remission, but with the initiation of mesalamine treatment, they had abdominal pain and bloody mucoid diarrhoea and/or elevation of white blood cell count, C-reactive protein level and erythrocyte sedimentation rate. Bacterial pathogens were excluded from the urine, throat and blood cultures, parasites with stool examination, viral pathogens with serology. Within 3–7 days after the mesalamine treatment had been stopped, the patients showed improvement of colitis symptoms and normalisation of white blood cell count, C-reactive protein level and erythrocyte sedimentation rate. Conclusion In this study mesalamine mimicked CD relapse in children with CD while tapering or after stopping steroid treatment. Awareness of this side effect of mesalamine could prevent a misdiagnosis of steroid dependency. PMID:26630041

New monoaromatic steroids in organic matter of the apocatagenesis zone

NASA Astrophysics Data System (ADS)

Kashirtsev, V. A.; Fomin, A. N.; Shevchenko, N. P.; Dolzhenko, K. V.

2016-08-01

According to the materials of geochemical study in the core of the ultradeep hole SV-27 of aromatic fractions of bitumoids of the Vilyui syneclise (East Siberia) by the method of chromatography-mass spectrometry, starting from the depth of >5000 m, four diastereomers of previously unknown hydrocarbons, which become predominant in the fraction at a depth of ˜6500 m, were distinguished. Similar hydrocarbons were found in organic matter of Upper Paleozoic rocks of the Kharaulakh anticlinorium in the Verkhoyansk folded area. According to the intense molecular ion m/z 366 and the character of the basic fragmental ions (m/z 238, 309, and 323), the major structure of the compounds studied was determined as 17-desmethyl-23-methylmonoaromatic steroid C27. The absence of such steroids in oil of the Vilyui syneclise shows that deep micro-oils did not participate in the formation of oil fringes of gas condensate deposits of the region.

Saliva as a tool for monitoring steroid, peptide and immune markers in sport and exercise science.

PubMed

Papacosta, Elena; Nassis, George P

2011-09-01

This paper discusses the use of saliva analysis as a tool for monitoring steroid, peptide, and immune markers of sports training. Salivary gland physiology, regarding the regulation and stimulation of saliva secretion, as well as methodological issues including saliva collection, storage and analysis are addressed in this paper. The effects of exercise on saliva composition are then considered. Exercise elicits changes in salivary levels of steroid hormones, immunoglobulins, antimicrobial proteins and enzymes. Cortisol, testosterone and dehydroepiandrosterone can be assessed in saliva, providing a non-invasive option to assess the catabolic and anabolic effects of exercise. Validation studies using blood and salivary measures of steroid hormones are addressed in this paper. Effects of acute exercise and training on salivary immunoglobulins (SIgA, SIgM, SIgG) and salivary antimicrobial proteins, including α-amylase, lysozyme and lactoferrin, are also discussed. Analysis of cortisol and testosterone in saliva may help detect the onset of non-functional overreaching and subsequently may help to prevent the development of overtraining syndrome. Assessment of salivary immunoglobulins and antimicrobial proteins has been shown to successfully represent the effects of exercise on mucosal immunity. Increases in SIgA and antimicrobial proteins concentration and/or secretion rate are associated with acute exercise whereas conversely, decreases have been reported in athletes over a training season leaving the athlete susceptible for upper respiratory tract infections. The measurement of physiological biomarkers in whole saliva can provide a significant tool for assessing the immunological and endocrinological status associated with exercise and training. Copyright © 2011 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

PubMed Central

Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

2016-01-01

Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

Changes of MK medium during storage of human cornea.

PubMed Central

Hasany, S M; Basu, P K

1987-01-01

By comparing the composition of McCarey-Kaufman (MK) medium before and after corneal storage we attempted to identify specific physiological changes in the medium as predictors of tissue damage. We also tried to determine if hydrocortisone (a lysosomal membrane stabiliser) added to the medium could reduce tissue damage during storage. Corneas (human and rabbit) were stored in the MK medium with and without hydrocortisone for 4 days at 4 degrees C. The water and nitrogen contents of the stored cornea were compared with those of the fresh cornea. The medium was analysed before and after corneal storage to determine the concentrations of glucose, protein, and amino acids as well as pH and osmolarity. Scanning electron microscopy (SEM) was used to estimate the degree of the corneal endothelial cell damage. The nitrogen contents and dry weights of the steroid treated and untreated stored corneas were similar to those of the fresh unstored cornea. The steroid treated cornea contained a lesser amount of water than the untreated cornea. The cornea stored in medium without steroid took up a greater amount of glucose from the medium than the cornea stored in medium with steroid. As compared with their concentrations in the fresh unused medium the concentrations of leucine, lysine, and glycine were lower and that of glutamic acid was higher in both the media used for corneal storage. However, the steroid treated storage medium as compared with the untreated storage medium had a greater reduction in the lowering of leucine, lysine, and glycine, and a lesser reduction in the increase of glutamic acid. Steroid treated medium also had a lesser amount of protein released from the stored cornea. Changes in the pH and osmolarity of the media before and after corneal storage were not remarkable. SEM showed that the endothelial cells of the cornea stored in the medium containing steroid were less damaged than those of the cornea stored in the medium without steroid. Images PMID:3620430

Current Knowledge on the Acute Regulation of Steroidogenesis.

PubMed

Selvaraj, Vimal; Stocco, Douglas M; Clark, Barbara J

2018-04-27

How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early was that acute regulation of steroid biosynthesis required swift and timely synthesis of a new protein whose role appeared to be involved in the delivery of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where the process of steroidogenesis begins. It was quickly learned that this transfer of cholesterol to the inner mitochondrial membrane was the regulated and rate limiting step in steroidogenesis. Following this observation, the quest for this putative regulator protein(s) began in earnest in the late 1950s. This review provides a history of this quest, the candidate proteins that arose over the years, and facts surrounding their rise or decline. Only two have persisted-Translocator Protein (TSPO) and the Steroidogenic Acute Regulatory Protein (StAR). We present a detailed summary of the work that has been published for each of these two proteins, the specific data that has appeared in support of their role in cholesterol transport and steroidogenesis, and the ensuing observations that have arisen in recent years that have refuted the role of TSPO in this process. We believe that the only viable candidate that has been shown to be indispensable is the StAR protein. Lastly, we provide our view on what may be the most important questions concerning the acute regulation of steroidogenesis that need to be asked in future.

77 FR 44456 - Classification of Two Steroids, Prostanozol

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-30

... by positive nitrogen balance and protein metabolism, resulting in increases in protein synthesis and... activity by means of nitrogen balance and androgenic activity based on weight changes of the ventral...

Expression of androgen-binding protein (ABP) in human cardiac myocytes.

PubMed

Schock, H W; Herbert, Z; Sigusch, H; Figulla, H R; Jirikowski, G F; Lotze, U

2006-04-01

Cardiomyocytes are known to be androgen targets. Changing systemic steroid levels are thought to be linked to various cardiac ailments, including dilated cardiomyopathy (DCM). The mode of action of gonadal steroid hormones on the human heart is unknown to date. In the present study, we used high-resolution immunocytochemistry on semithin sections (1 microm thick), IN SITU hybridization, and mass spectrometry to investigate the expression of androgen-binding protein (ABP) in human myocardial biopsies taken from male patients with DCM. We observed distinct cytoplasmic ABP immunoreactivity in a fraction of the myocytes. IN SITU hybridization with synthetic oligonucleotide probes revealed specific hybridization signals in these cells. A portion of the ABP-positive cells contained immunostaining for androgen receptor. With SELDI TOF mass spectrometry of affinity purified tissue extracts of human myocardium, we confirmed the presence of a 50 kDa protein similar to ABP. Our observations provide evidence of an intrinsic expression of ABP in human heart. ABP may be secreted from myocytes in a paracrine manner perhaps to influence the bioavailabity of gonadal steroids in myocardium.

Ophirapstanol trisulfate, a new biologically active steroid sulfate from the deep water marine sponge Topsentia ophiraphidites.

PubMed

Gunasekera, S P; Sennett, S H; Kelly-Borges, M; Bryant, R W

1994-12-01

Ophirapstanol trisulfate [1], a new steroid trisulfate related to sokotrasterol trisulfate was isolated from a deep water marine sponge Topsentia ophiraphidites. Compound 1 exhibited significant inhibition in the guanosine diphosphate/G-protein RAS exchange assay. The structure elucidation of 1 and ophirapstanol [2] by nmr spectroscopy is described.

Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression.

PubMed

Draisci, Rosa; Montesissa, Clara; Santamaria, Barbara; D'Ambrosio, Chiara; Ferretti, Giovanni; Merlanti, Roberta; Ferranti, Carolina; De Liguoro, Marco; Cartoni, Claudia; Pistarino, Erika; Ferrara, Lino; Tiso, Micaela; Scaloni, Andrea; Cosulich, M Elisabetta

2007-09-01

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-Q-MS/MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone alpha- and beta-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE, MALDI-TOF-MS and muLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasma samples collected before treatment, was found upregulated even 36 h after hormone treatment. Extensive mass mapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed by Western blot analysis confirmed a significant and time dependent increase of this ApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine.

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

1990-02-20

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

Human hydroxysteroid dehydrogenases and pre-receptor regulation: Insights into inhibitor design and evaluation

PubMed Central

Penning, Trevor M.

2011-01-01

Hydroxysteroid dehydrogenases (HSDs) represent a major class of NAD(P)(H) dependent steroid hormone oxidoreductases involved in the pre-receptor regulation of hormone action. This is achieved by HSDs working in pairs so that they can interconvert ketosteroids with hydroxysteroids resulting in a change in ligand potency for nuclear receptors. HSDs belong to two protein superfamilies the aldo-keto reductases and the short-chain dehydrogenase/reductases. In humans, many of the important enzymes have been thoroughly characterized including the elucidation of their three-dimensional structures. Because these enzymes play fundamental roles in steroid hormone action they can be considered to be drug targets for a variety of steroid driven diseases: e.g. metabolic syndrome and obesity, inflammation, and hormone dependent malignancies of the endometrium, prostate and breast. This article will review how fundamental knowledge of these enzymes can be exploited in the development of isoform specific HSD inhibitors from both protein superfamilies. PMID:21272640

Profiling intact steroid sulfates and unconjugated steroids in biological fluids by liquid chromatography-tandem mass spectrometry (LC-MS-MS).

PubMed

Galuska, Christina E; Hartmann, Michaela F; Sánchez-Guijo, Alberto; Bakhaus, Katharina; Geyer, Joachim; Schuler, Gerhard; Zimmer, Klaus-Peter; Wudy, Stefan A

2013-07-07

Within the combined DFG research project "Sulfated Steroids in Reproduction" an analytical method was needed for determining sulfated and unconjugated steroids with highest specificity out of different biological matrices such as aqueous solution, cell lysate and serum. With regard to this analytical challenge, LC-MS-MS presents the technique of choice because it permits (1) analysis of the intact steroid conjugate, (2) allows for simultaneous determination of multiple analytes (profiling, targeted metabolomics approach) and (3) is independent of phenomena such as cross-reactivity. Sample work up consisted of incubation of sample with internal standards (deuterium labeled steroids) followed by solid phase extraction. Only serum samples required a protein precipitation step prior to solid phase extraction. The extract was divided in two parts: six steroid sulfates (E1S, E2S, AS, 16-OH-DHEAS, PREGS, DHEAS) were analyzed by C18aQ-ESI-MS-MS in negative ion mode and eleven unconjugated steroids (E3, 16-OH-DHEA, E1, E2, (4)A, DHEA, T, 17-OH-PREG, Prog, An, PREG) were analyzed by C18-APCI-MS-MS in positive ion mode. For steroid sulfates, we found high sensitivities with LoQ values ranging from 0.08 to 1 ng mL(-1). Unconjugated steroids showed LoQ values between 0.5 and 10 ng mL(-1). Calibration plots showed excellent linearity. Mean intra- and inter-assay CVs were 2.4% for steroid sulfates and 6.4% for unconjugated steroids. Accuracy - determined in a two-level spike experiment - showed mean relative errors of 5.9% for steroid sulfates and 6.1% for unconjugated steroids. In summary, we describe a novel LC-MS-MS procedure capable of profiling six steroid sulfates and eleven unconjugated steroids from various biological matrices.

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

PubMed Central

Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

2014-01-01

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

Neurosecretory cells of the amygdaloid complex during estrous cycle.

PubMed

Akhmadeev, A V; Kalimullina, L B

2005-02-01

Ultrastructure of neurosecretory cells of the dorsomedial nucleus of the cerebral amygdaloid complex (one of the main zones of sexual dimorphism) was studied in different phases of the estrous cycle. The characteristics of the "light" and "dark" cells change depending on the concentrations of sex steroids during estrus and metestrus.

Promotion of bone growth by dietary soy protein isolate: Comparision with dietary casein, whey hydrolysate and rice protein isolate in growing female rats

USDA-ARS?s Scientific Manuscript database

The effects of different dietary protein sources(casein (CAS), soy protein isolate (SPI), whey protein hydrolysate (WPH) and rice protein isolate (RPI)) on bone were studied in intact growing female rats and in ovarectomized (OVX) rats showing sex steroid deficiency-induced bone loss. In addition, S...

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators

PubMed Central

Redfern, Andrew D.; Colley, Shane M.; Beveridge, Dianne J.; Ikeda, Naoya; Epis, Michael R.; Li, Xia; Foulds, Charles E.; Stuart, Lisa M.; Barker, Andrew; Russell, Victoria J.; Ramsay, Kerry; Kobelke, Simon J.; Li, Xiaotao; Hatchell, Esme C.; Payne, Christine; Giles, Keith M.; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B.; O’Malley, Bert W.; Leedman, Peter J.

2013-01-01

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing. PMID:23550157

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

PubMed

Redfern, Andrew D; Colley, Shane M; Beveridge, Dianne J; Ikeda, Naoya; Epis, Michael R; Li, Xia; Foulds, Charles E; Stuart, Lisa M; Barker, Andrew; Russell, Victoria J; Ramsay, Kerry; Kobelke, Simon J; Li, Xiaotao; Hatchell, Esme C; Payne, Christine; Giles, Keith M; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B; O'Malley, Bert W; Leedman, Peter J

2013-04-16

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

Three novel degraded steroids from cultures of the Basidiomycete Antrodiella albocinnamomea.

PubMed

Chen, Zi-Ming; Yang, Xiao-Yan; Fan, Qiong-Ying; Li, Zheng-Hui; Wei, Kun; Chen, He-Ping; Feng, Tao; Liu, Ji-Kai

2014-09-01

Three novel degraded steroids, named albocisterols A-C (1-3), have been isolated from cultures of Antrodiella albocinnamomea. Their structures were defined by comprehensive spectroscopic analysis and single crystal X-ray crystallography. The mixture of compounds 2 and 3 exhibited significant inhibitory activities against protein tyrosine phosphatase 1B (PTP1B). Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of sex steroids on indices of protein turnover in rainbow trout (Oncorhynchus mykiss) white muscle

USDA-ARS?s Scientific Manuscript database

Effects of 17-estradiol (E2), testosterone, and 5a-dihydrotestosterone (DHT) on protein turnover and proteolytic gene expression were determined in rainbow trout (Oncorhynchus mykiss) primary myocytes and white muscle tissue. E2 reduced rates of protein synthesis and increased rates of protein degr...

De novo steroid biosynthesis in human prostate cell lines and biopsies.

PubMed

Sakai, Monica; Martinez-Arguelles, Daniel B; Aprikian, Armen G; Magliocco, Anthony M; Papadopoulos, Vassilios

2016-05-01

Intratumoral androgen formation may be a factor in the development of prostate cancer (PCa), particularly castration-resistant prostate cancer (CRPC). To evaluate the ability of the human prostate to synthesize de novo steroids, we examined the expression of key enzymes and proteins involved in steroid biosynthesis and metabolism. Using TissueScan™ Cancer qPCR Arrays and quantitative RT-PCR, we performed comparative gene expression analyses between various prostate cell lines and biopsies, including normal, hyperplastic, cancerous, and androgen-deprived prostate cells lines, as well as normal, benign prostate hyperplasia (BPH), PCa, and CRPC human specimens. These studies were complemented with steroid biosynthesis studies in normal and BPH cells. Normal human prostate WPMY-1 and WPE1-NA22, benign prostate hyperplasia BPH-1, and cancer PC-3, LNCaP, and VCaP cell lines, as well as normal, BPH, PCa, and CRPC specimens, were used. Although all cell lines express mRNA encoding for hydroxymethylglutaryl-CoA reductase (HMGCR), the mitochondrial translocator protein TSPO and cholesterol side chain cleavage enzyme CYP11A1 were only observed in WPMY-1, BPH-1, and LNCaP cells. HSD3B1, HSD3B2, and CYP17A1 are involved in androgen formation and were not found in most cell lines. WPE1-NA22 and BPH-1 cells were unable to synthesize de novo steroids from mevalonate. Moreover, androgen-deprived cells did not have alterations in the expression of enzymes that could lead to de novo steroid formation. All prostate specimens expressed TSPO and CYP11A1. HSD3B1/2, CYP17A1, HSD17B5, and CYP19A1 mRNA expression was distinct to the profile observed in cells lines. The majority of BPH (90.9%) and PCa (83.1%) specimens contained CYP17A1, compared to control (normal) specimens (46.7%). BPH (82%), PCa (59%), normal (40%), and CRPC (34%) specimens expressed the four key enzymes that metabolize cholesterol to androgens. These studies question the use of prostate cell lines to study steroid biosynthesis and demonstrate that human prostate samples contain transcripts encoding for key steroidogenic enzymes and proteins indicating that they have the potential to synthesize de novo steroids. We propose CYP17A1 as a candidate enzyme that can be used for patient stratification and treatment in BPH and PCa. © 2016 Wiley Periodicals, Inc.

Regulation of extracellular matrix vesicles via rapid responses to steroid hormones during endochondral bone formation.

PubMed

Asmussen, Niels; Lin, Zhao; McClure, Michael J; Schwartz, Zvi; Boyan, Barbara D

2017-12-09

Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH) 2 D 3 ] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH) 2 D 3 ] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH) 2 D 3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs. Copyright © 2017. Published by Elsevier Inc.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM--role of satellite cells in anabolic steroid-induced muscle growth in feedlot steers.

PubMed

Dayton, W R; White, M E

2014-01-01

Both androgenic and estrogenic steroids are widely used as growth promoters in feedlot steers because they significantly enhance feed efficiency, rate of gain, and muscle growth. However, despite their widespread use relatively little is known about the biological mechanism by which androgenic and estrogenic steroids enhance rate and efficiency of muscle growth in cattle. Treatment of feedlot steers with a combined estradiol (E2) and trenbolone acetate (TBA) implant results in an increased number of muscle satellite cells, increased expression of IGF-1 mRNA in muscle tissue, and increased levels of circulating IGF-1. Similarly, treatment of bovine satellite cell (BSC) cultures with either TBA or E2 results in increased expression of IGF-1 mRNA, increased rates of proliferation and protein synthesis, and decreased rates of protein degradation. Effects of E2 on BSC are mediated at least in part through the classical E2 receptor, estrogen receptor-α (ESR1), the IGF-1 receptor (IGFR1), and the G protein-coupled estrogen receptor-1 (GPER-1), formerly known as G protein-coupled receptor-30 (GPR30). The effects of TBA appear to be primarily mediated through the androgen receptor. Based on current research results, it is becoming clear that anabolic steroid-enhanced bovine muscle growth involves a complex interaction of numerous pathways and receptors. Consequently, additional in vivo and in vitro studies are necessary to understand the mechanisms involved in this complex process. The fundamental information generated by this research will help in developing future, safe, and effective strategies to increase rate and efficiency of muscle growth in beef cattle.

The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.

PubMed

Estabrook, R W; Shet, M S; Faulkner, K; Fisher, C W

1996-11-01

A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.

Covalent modification of proteins by ligands of steroid hormone receptors.

PubMed

Takahashi, N; Breitman, T R

1992-11-15

Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in HL-60 cells were labeled by 17 beta-[3H]estradiol (E2), [3H]progesterone (Pg), 1 alpha,25-dihydroxy[3H]vitamin D3 [1,25(OH)2D3], [125I]triiodothyronine (T3), [125I]thyroxine (T4), and [3H]prostaglandin E2 (PGE2). All of these hormones, except PGE2, are ligands of the steroid hormone receptor family. Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2. In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg. Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein. These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3. Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands. Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand. Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4. These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases. 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000. However, these proteins had pI values different from those of RI or RII. These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins.

Autoantibodies to recombinant lipocortin-1 in rheumatoid arthritis and systemic lupus erythematosus.

PubMed Central

Goulding, N J; Podgorski, M R; Hall, N D; Flower, R J; Browning, J L; Pepinsky, R B; Maddison, P J

1989-01-01

Corticosteroids may mediate some of their anti-inflammatory effects via induction of a specific 38 kD protein, lipocortin-1. Autoantibodies to lipocortin-1 were measured by enzyme linked immunosorbent assay (ELISA) in 90 healthy subjects and in 63 patients with rheumatoid arthritis (RA), 36 with systemic lupus erythematosus (SLE), 26 with polymyalgia rheumatica, and 13 with chronic airways disease. Sixteen patients with RA receiving prolonged, high steroid doses (prednisolone greater than 7.5 mg/day) had raised IgM antilipocortin-1 levels, while 19 patients with RA untreated with steroids had normal levels. This association was independent of disease activity. In SLE, raised antilipocortin-1 levels were associated with active disease and were independent of steroid treatment. Antilipocortin-1 antibody levels were not raised in patients with polymyalgia rheumatica and chronic airways disease. Thus steroid treatment alone appears insufficient to induce antilipocortin-1 antibodies, unless an underlying autoimmune state is also present. In RA, antilipocortin-1 antibodies may impair anti-inflammatory actions of steroids and render some patients 'steroid resistant'. PMID:2554826

Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium.

PubMed

Gerber, Adrian; Kleser, Michael; Biedendieck, Rebekka; Bernhardt, Rita; Hannemann, Frank

2015-07-29

Cholesterol, the precursor of all steroid hormones, is the most abundant steroid in vertebrates and exhibits highly hydrophobic properties, rendering it a difficult substrate for aqueous microbial biotransformations. In the present study, we developed a Bacillus megaterium based whole-cell system that allows the side-chain cleavage of this sterol and investigated the underlying physiological basis of the biocatalysis. CYP11A1, the side-chain cleaving cytochrome P450, was recombinantly expressed in the Gram-positive soil bacterium B. megaterium combined with the required electron transfer proteins. By applying a mixture of 2-hydroxypropyl-β-cyclodextrin and Quillaja saponin as solubilizing agents, the zoosterols cholesterol and 7-dehydrocholesterol, as well as the phytosterol β-sitosterol could be efficiently converted to pregnenolone or 7-dehydropregnenolone. Fluorescence-microscopic analysis revealed that cholesterol accumulates in the carbon and energy storage-serving poly(3-hydroxybutyrate) (PHB) bodies and that the membrane proteins CYP11A1 and its redox partner adrenodoxin reductase (AdR) are likewise localized to their surrounding phospholipid/protein monolayer. The capacity to store cholesterol was absent in a mutant strain devoid of the PHB-producing polymerase subunit PhaC, resulting in a drastically decreased cholesterol conversion rate, while no effect on the expression of the recombinant proteins could be observed. We established a whole-cell system based on B. megaterium, which enables the conversion of the steroid hormone precursor cholesterol to pregnenolone in substantial quantities. We demonstrate that the microorganism's PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer, are crucial for the high conversion rate by serving as substrate storage. This microbial system opens the way for an industrial conversion of the abundantly available cholesterol to any type of steroid hormones, which represent one of the biggest groups of drugs for the treatment of a wide variety of diseases.

An In-Silico Investigation of Phytochemicals as Antiviral Agents Against Dengue Fever.

PubMed

Powers, Chelsea N; Setzer, William N

2016-01-01

A virtual screening analysis of our library of phytochemical structures with dengue virus protein targets has been carried out using a molecular docking approach. A total of 2194 plant-derived secondary metabolites have been docked. This molecule set comprised of 290 alkaloids (68 indole alkaloids, 153 isoquinoline alkaloids, 5 quinoline alkaloids, 13 piperidine alkaloids, 14 steroidal alkaloids, and 37 miscellaneous alkaloids), 678 terpenoids (47 monoterpenoids, 169 sesquiterpenoids, 265 diterpenoids, 81 steroids, and 96 triterpenoids), 20 aurones, 81 chalcones, 349 flavonoids, 120 isoflavonoids, 74 lignans, 58 stilbenoids, 169 miscellaneous polyphenolic compounds, 100 coumarins, 28 xanthones, 67 quinones, and 160 miscellaneous phytochemicals. Dengue virus protein targets examined included dengue virus protease (NS2B-NS3pro), helicase (NS3 helicase), methyltransferase (MTase), RNA-dependent RNA polymerase (RdRp), and the dengue virus envelope protein. Polyphenolic compounds, flavonoids, chalcones, and other phenolics were the most numerous of the strongly docking ligands for dengue virus protein targets.

GPR30: A G protein-coupled receptor for estrogen.

PubMed

Prossnitz, Eric R; Arterburn, Jeffrey B; Sklar, Larry A

2007-02-01

Estrogen is a critical steroid in human physiology exerting its effect both at the transcriptional level as well as at the level of rapid intracellular signaling through second messengers. Many of estrogen's transcriptional effects have long been known to be mediated through classical nuclear steroid receptors but recent studies also demonstrate the existence of a 7-transmembrane G protein-coupled receptor, GPR30 that responds to estrogen with rapid cellular signaling. There is currently controversy over the ability of classical estrogen receptors to recapitulate GPR30-mediated signaling mechanisms and vice versa. This article will summarize recent literature and address the relationship between GPR30 and conventional estrogen receptor signaling.

G protein-coupled receptor 30 down-regulates cofactor expression and interferes with the transcriptional activity of glucocorticoid.

PubMed

Ylikomi, Timo; Vienonen, Annika; Ahola, Tytti M

2004-11-01

G protein-coupled receptor 30 (GPR30) has previously been described to be important in steroid-mediated growth and to inhibit cell proliferation. Here we investigated whether the effect of GPR30 on cell growth is dependent on steroid hormone receptors. We stably introduced GPR30 in immortalized normal mammary epithelial (HME) cells using retroviruses for gene delivery. GPR30 inhibited the growth and proliferation of the cells. They expressed glucocorticoid receptor, but not estrogen or progesterone receptor. GPR30 down-regulated the expression of cofactor transcription intermediary factor 2 (TIF2) analyzed using quantitative RT-PCR analysis, and also diminished the expression of TIF2 at protein level analyzed by Western blotting using nuclear extracts from mammary epithelial cells. When HME cells were transiently transfected with the glucocorticoid response element MMTV-luc reporter plasmid, stable expression of GPR30 resulted in the abolition of ligand-induced transactivation of the promoter. In COS cells, transient transfection of GPR30 with glucocorticoid receptor alpha resulted in an abrogation of the MMTV-luc and GRE-luc reporter activities induced by dexamethasone. The results suggest a novel mechanism by which membrane-initiated signaling interferes with steroid signaling.

Neurosteroidogenesis today: Novel targets for neuroactive steroid synthesis and action and their relevance for translational research

PubMed Central

Porcu, Patrizia; Barron, Anna M.; Frye, Cheryl Anne; Walf, Alicia A.; Yang, Song-Yu; He, Xue-Ying; Morrow, A. Leslie; Panzica, Gian Carlo; Melcangi, Roberto C.

2015-01-01

Neuroactive steroids are endogenous neuromodulators synthesised in the brain that rapidly alter neuronal excitability by binding to membrane receptors, in addition to the regulation of gene expression via intracellular steroid receptors. Neuroactive steroids induce potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the γ-amino-butyric type A (GABAA) receptor. They also exert neuroprotective, neurotrophic and antiapoptotic effects in several animal models of neurodegenerative diseases. Neuroactive steroids regulate many physiological functions such as stress response, puberty, ovarian cycle, pregnancy and reward. Their levels are altered in several neuropsychiatric and neurologic diseases and both preclinical and clinical studies emphasise a therapeutic potential of neuroactive steroids for these diseases, whereby symptomatology ameliorates upon restoration of neuroactive steroid concentrations. However, direct administration of neuroactive steroids has several challenges, including pharmacokinetics, low bioavailability, addiction potential, safety and tolerability that limit its therapeutic use. Therefore, modulation of neurosteroidogenesis to restore the altered endogenous neuroactive steroid tone may represent a better therapeutic approach. This review summarizes recent approaches that target the neuroactive steroid biosynthetic pathway at different levels in order to promote neurosteroidogenesis. These include modulation of neurosteroidogenesis through ligands of the translocator protein 18 kDa (TSPO), and the pregnane xenobiotic receptor (PXR), as well as targeting of specific neurosteroidogenic enzymes like 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) or P450 side chain cleavage (P450scc). Enhanced neurosteroidogenesis through these targets may be beneficial for neurodegenerative diseases such as Alzheimer's disease and age-related dementia, but also for neuropsychiatric diseases, including alcohol use disorders. PMID:26681259

Sustained Local Release of Methylprednisolone From a Thiol-Acrylate Poly(Ethylene Glycol) Hydrogel for Treating Chronic Compressive Radicular Pain.

PubMed

Slotkin, Jonathan R; Ness, Jennifer K; Snyder, Kristin M; Skiles, Amanda A; Woodard, Eric J; OʼShea, Timothy; Layer, Rick T; Aimetti, Alex A; Toms, Steven A; Langer, Robert; Tapinos, Nikos

2016-04-01

A preclinical animal model of chronic ligation of the sciatic nerve was used to compare the effectiveness of a slow-release hydrogel carrying methylprednisolone to methylprednisolone injection alone, which simulates the current standard of care for chronic compressive radiculopathy (CR). To extend the short-term benefits of steroid injections by using a nonswelling, biodegradable hydrogel as carrier to locally release methylprednisolone in a regulated and sustained way at the site of nerve compression. CR affects millions worldwide annually, and is a cause of costly disability with significant societal impact. Currently, a leading nonsurgical therapy involves epidural injection of steroids to temporarily alleviate the pain associated with CR. However, an effective way to extend the short-term effect of steroid treatment to address the chronic component of CR does not exist. We induced chronic compression injury of the sciatic nerves of rats by permanent ligation. Forty-eight hours later we injected our methylprednisolone infused hydrogel and assessed the effectiveness of our treatment for 4 weeks. We quantified mechanical hyperalgesia using a Dynamic Plantar Aesthesiometer (Ugo Basile, Stoelting Co., IL, USA), whereas gait analysis was conducted using the Catwalk automated gait analysis platform (Noldus, Leesburg, VA, USA). Macrophage staining was performed with immunohistochemistry and quantification of monocyte chemoattractant protein-1 in sciatic nerve lysates was performed with multiplex immunoassay using a SECTOR Imager 2400A (Meso Scale Discovery, Rockville, MA, USA). We demonstrate that using the hydrogel to deliver methylprednisolone results in significant (P < 0.05) reduction of hyperalgesia and improvement in the gait pattern of animals with chronic lesions as compared with animals treated with steroid alone. In addition, animals treated with hydrogel plus steroid showed significant reduction in the number of infiltrating macrophages at the sciatic nerve and reduced expression of the neuroinflammatory chemokine monocyte chemoattractant protein-1 (P < 0.05). Use of hydrogels as carriers for sustained local release of steroids provides significantly better control of pain in an animal model of chronic CR. Our steroid-infused hydrogel could be an effective extender of the short-term benefits of epidural steroid injections for patients with chronic compression-induced radicular pain. N/A.

5β-Reduced Steroids and Human Δ4-3-Ketosteroid 5β-Reductase (AKR1D1)

PubMed Central

Chen, Mo; Penning, Trevor M.

2014-01-01

5β-Reduced steroids are non-planar steroids that have 90° bend in their structure to create an A/B cis-ring junction. This novel property is required for bile-acids to act as emulsifiers, but in addition 5β-reduced steroids have remarkable physiology and may act as potent tocolytic agents, endogenous cardiac glycosides, neurosteroids, and can act as ligands for orphan and membrane bound receptors. In humans there is only a single 5β-reductase gene AKR1D1, which encodes Δ4-3-ketosteroid-5β-reductase (AKR1D1). This enzyme is a member of the aldoketo reductase superfamily, but possesses an altered catalytic tetrad, in which Glu120 replaces the conserved His residue. This predominant liver enzyme generates all 5β-dihydrosteroids in the C19–C27 steroid series. Mutations exist in the AKR1D1 gene, which result in loss of protein stability and are causative in bile-acid deficiency. PMID:24513054

Inheritance of steroid-independent male sexual behavior in male offspring of B6D2F1 mice.

PubMed

McInnis, Christine M; Bonthuis, Paul J; Rissman, Emilie F; Park, Jin Ho

2016-04-01

The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrates steroid-independent male sexual behavior (identified as "maters"), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones. Recent studies have revealed several proteins that play a significant factor in regulating steroid-independent male sexual behavior in B6D2F1 male mice, including amyloid precursor protein (APP), tau, and synaptophysin. The specific goals of our study were to determine whether steroid-independent male sexual behavior was a heritable trait by determining if it was dependent upon the behavioral phenotype of the B6D2F1 sire, and whether the differential expression of APP, tau, and synaptophysin in the medial preoptic area found in the B6D2F1 sires that did and did not mate after gonadectomy was similar to those found in their male offspring. After adult B6D2F1 male mice were bred with C57BL/6J female mice, they and their male offspring (BXB1) were orchidectomized and identified as either maters or "non-maters". A significant proportion of the BXB1 maters was sired only from B6D2F1 maters, indicating that the steroid-independent male sexual behavior behavioral phenotype of the B6D2F1 hybrid males, when crossed with C57BL/6J female mice, is inherited by their male offspring. Additionally, APP, tau, and synaptophysin were elevated in in the medial preoptic area in both the B6D2F1 and BXB1 maters relative to the B6D2F1 and BXB1 non-maters, respectively, suggesting a potential genetic mechanism for the inheritance of steroid-independent male sexual behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

Inheritance of steroid-independent male sexual behavior in male offspring of B6D2F1 mice

PubMed Central

McInnis, Christine M.; Bonthuis, Paul J.; Rissman, Emilie F.; Park, Jin Ho

2016-01-01

The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrate steroid-independent male sexual behavior (identified as “maters”), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones. Recent studies have revealed several proteins that play a significant factor in regulating steroid-independent male sexual behavior in B6D2F1 male mice, including amyloid precursor protein (APP), tau, and synaptophysin. The specific goals of our study were to determine whether steroid-independent male sexual behavior was a heritable trait by determining if it was dependent upon the behavioral phenotype of the B6D2F1 sire, and whether the differential expression of APP, tau, and synaptophysin in the medial preoptic area found in the B6D2F1 sires that did and did not mate after gonadectomy was similar to those found in their male offspring. After adult B6D2F1 male mice were bred with C57BL/6J female mice, they and their male offspring (BXB1) were orchidectomized and identified as either maters or “non-maters.” A significant proportion of the BXB1 maters were sired only from B6D2F1 maters, indicating that the steroid-independent male sexual behavior behavioral phenotype of the B6D2F1 hybrid males, when crossed with C57BL/6J female mice, is inherited by their male offspring. Additionally, APP, tau, and synaptophysin were elevated in in the medial preoptic area in both the B6D2F1 and BXB1 maters relative to the B6D2F1 and BXB1 non-maters, respectively, suggesting a potential genetic mechanism for the inheritance of steroid-independent male sexual behavior. PMID:26940434

Steroid receptor RNA activator (SRA1): unusual bifaceted gene products with suspected relevance to breast cancer

PubMed Central

Leygue, Etienne

2007-01-01

The steroid receptor RNA activator (SRA) is a unique modulator of steroid receptor transcriptional activity, as it is able to mediate its coregulatory effects as a RNA molecule. Recent findings, however, have painted a more complex picture of the SRA gene (SRA1) products. Indeed, even though SRA was initially thought to be noncoding, several RNA isoforms have now been found to encode an endogenous protein (SRAP), which is well conserved among Chordata. Although the function of SRAP remains largely unknown, it has been proposed that, much like its corresponding RNA, the protein itself might regulate estrogen and androgen receptor signaling pathways. As such, data suggest that both SRA and SRAP might participate in the mechanisms underlying breast, as well as prostate tumorigenesis. This review summarizes the published literature dealing with these two faces of the SRA gene products and underscores the relevance of this bifaceted system to breast cancer development. PMID:17710122

A novel progesterone receptor membrane component (PGRMC) in the human and swine parasite Taenia solium: implications to the host-parasite relationship.

PubMed

Aguilar-Díaz, Hugo; Nava-Castro, Karen E; Escobedo, Galileo; Domínguez-Ramírez, Lenin; García-Varela, Martín; Del Río-Araiza, Víctor H; Palacios-Arreola, Margarita I; Morales-Montor, Jorge

2018-03-09

We have previously reported that progesterone (P 4 ) has a direct in vitro effect on the scolex evagination and growth of Taenia solium cysticerci. Here, we explored the hypothesis that the P 4 direct effect on T. solium might be mediated by a novel steroid-binding parasite protein. By way of using immunofluorescent confocal microscopy, flow cytometry analysis, double-dimension electrophoresis analysis, and sequencing the corresponding protein spot, we detected a novel PGRMC in T. solium. Molecular modeling studies accompanied by computer docking using the sequenced protein, together with phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is from parasite origin. Our results show that P 4 in vitro increases parasite evagination and scolex size. Using immunofluorescent confocal microscopy, we detected that parasite cells showed expression of a P 4 -binding like protein exclusively located at the cysticercus subtegumental tissue. Presence of the P 4 -binding protein in cyst cells was also confirmed by flow cytometry. Double-dimension electrophoresis analysis, followed by sequencing the corresponding protein spot, revealed a protein that was previously reported in the T. solium genome belonging to a membrane-associated progesterone receptor component (PGRMC). Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus, both of them being nested within a cluster including similar proteins present in platyhelminths such as Schistocephalus solidus and Schistosoma haematobium. Progesterone may directly act upon T. solium cysticerci probably by binding to PGRMC. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.

Additive Effect of Oral Steroid with Topical Nonsteroidal Anti-inflammatory Drug for Preventing Cystoid Macular Edema after Cataract Surgery in Patients with Epiretinal Membrane

PubMed Central

Kim, Seonghwan; Wee, Won Ryang

2017-01-01

Purpose To investigate the additive effect of oral steroid with topical nonsteroidal anti-inflammatory drug (NSAID) on cystoid macular edema (CME) in patients with epiretinal membrane (ERM) after cataract surgery. Methods Medical records of subjects who underwent uneventful cataract surgery (n = 1,349) were retrospectively reviewed; among these patients, those with pre-existing ERM (n = 81) were included. Patients were divided into two groups: one group had postoperative administration of oral steroid for 1 week (n = 45) and the other group did not have oral steroid administration (n = 36). Changes in macular thickness and incidence of CME were compared in both groups. Topical NSAIDs were administered in both groups for 1 month postoperatively. Definite CME and probable CME were defined by changes in retinal contour with or without cystoid changes. Change in central macular thickness of more than three standard deviations (≥90.17 µm) was defined as possible CME. Macular thickness was measured at 1 month after the operation by optical coherence tomography. Results The incidence of definite, probable, and possible CME were 2.22%, 4.44%, and 8.89% with the use of steroid and 2.78%, 5.56%, and 8.33% without steroid, respectively (p = 0.694, p = 0.603, and p = 0.625), and regardless of treatment group, the incidences in these patients were higher compared to incidences in whole subjects (1.26%, 2.30%, and 4.32%; p = 0.048, p = 0.032, and p = 0.038, respectively). The differences in macular thickness were not statistically different between the two groups. Average changes of central foveal thickness in 3 mm and 6 mm zone were 29.29 µm, 35.93 µm, and 38.02 µm with the use of steroid and 32.25 µm, 44.08 µm, and 45.39 µm without steroid (p = 0.747, p = 0.148, and p = 0.077, respectively). Conclusions This study suggests that administration of oral steroid may not have a synergistic effect in reduction of CME and retinal thickness in patients with pre-existing ERM after cataract surgery, when topical NSAIDs are applied. PMID:28913997

Purification, characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata.

PubMed

Feng, Bing; Hu, Wei; Ma, Bai-ping; Wang, Yong-ze; Huang, Hong-ze; Wang, Sheng-qi; Qian, Xiao-hong

2007-10-01

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50 degrees C, pH 4, and specific activity of 12.34 U mg of total protein(-1) under the conditions, using diosgenin-3-O-alpha-L-rhamnopyranosyl(1-->4)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin.

Steroids

NASA Astrophysics Data System (ADS)

Frey, Felix J.; Frey, Brigitte M.; Benet, Leslie Z.

If a radioimmunoassay, a protein binding method, or a colorimetric assay for the assessment of a steroid level is replaced by high performance liquid chromatography (HPLC), the cost for the determination of a steroid level increases at least initially because one must acquire the new HPLC equipment. Therefore, if an older method provides the same results as the new, "advanced" HPLC method, the only advantage resulting from the introduction of a high performance chromatographic assay is that gained by the manufacturer in terms of greater sales. Thus, justification for the assessment of steroids by HPLC is only obtained if the quality and/or quantity of information gained is significantly increased as compared to that provided by the conventional methods. But this evidential relation, that more and better information justifies a higher price in any case, is no longer true in health care, with the birth some years ago of the categoric imperative for the reduction of costs in the medical sector. That is, each new technology introduced for health maintenance should demonstrate at least a stabilizing impact on total medical expenditures. Therefore, after reviewing the presently available HPLC methods for the clinically important steroids, we will consider whether HPLC analyses for these steroids can be recommended without violating this vox populi.

Adhesive blood microsampling systems for steroid measurement via LC-MS/MS in the rat.

PubMed

Heussner, Kirsten; Rauh, Manfred; Cordasic, Nada; Menendez-Castro, Carlos; Huebner, Hanna; Ruebner, Matthias; Schmidt, Marius; Hartner, Andrea; Rascher, Wolfgang; Fahlbusch, Fabian B

2017-04-01

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) allows for the direct analysis of multiple hormones in a single probe with minimal sample volume. Rodent-based animal studies strongly rely on microsampling, such as the dry blood spot (DBS) method. However, DBS suffers the drawback of hematocrit-dependence (non-volumetric). Hence, novel volumetric microsampling techniques were introduced recently, allowing sampling of fixed accurate volumes. We compared these methods for steroid analysis in the rat to improve inter-system comparability. We analyzed steroid levels in blood using the absorptive microsampling devices Whatman® 903 Protein Saver Cards, Noviplex™ Plasma Prep Cards and the Mitra™ Microsampling device and compared the obtained results to the respective EDTA plasma levels. Quantitative steroid analysis was performed via LC-MS/MS. For the determination of the plasma volume factor for each steroid, their levels in pooled blood samples from each human adults and rats (18weeks) were compared and the transferability of these factors was evaluated in a new set of juvenile (21days) and adult (18weeks) rats. Hematocrit was determined concomitantly. Using these approaches, we were unable to apply one single volume factor for each steroid. Instead, plasma volume factors had to be adjusted for the recovery rate of each steroid and device individually. The tested microsampling systems did not allow the use of one single volume factor for adult and juvenile rats based on an unexpectedly strong hematocrit-dependency and other steroid specific (pre-analytic) factors. Our study provides correction factors for LC-MS/MS steroid analysis of volumetric and non-volumetric microsampling systems in comparison to plasma. It argues for thorough analysis of chromatographic effects before the use of novel volumetric systems for steroid analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Common structural features of cholesterol binding sites in crystallized soluble proteins

PubMed Central

Bukiya, Anna N.; Dopico, Alejandro M.

2017-01-01

Cholesterol-protein interactions are essential for the architectural organization of cell membranes and for lipid metabolism. While cholesterol-sensing motifs in transmembrane proteins have been identified, little is known about cholesterol recognition by soluble proteins. We reviewed the structural characteristics of binding sites for cholesterol and cholesterol sulfate from crystallographic structures available in the Protein Data Bank. This analysis unveiled key features of cholesterol-binding sites that are present in either all or the majority of sites: i) the cholesterol molecule is generally positioned between protein domains that have an organized secondary structure; ii) the cholesterol hydroxyl/sulfo group is often partnered by Asn, Gln, and/or Tyr, while the hydrophobic part of cholesterol interacts with Leu, Ile, Val, and/or Phe; iii) cholesterol hydrogen-bonding partners are often found on α-helices, while amino acids that interact with cholesterol’s hydrophobic core have a slight preference for β-strands and secondary structure-lacking protein areas; iv) the steroid’s C21 and C26 constitute the “hot spots” most often seen for steroid-protein hydrophobic interactions; v) common “cold spots” are C8–C10, C13, and C17, at which contacts with the proteins were not detected. Several common features we identified for soluble protein-steroid interaction appear evolutionarily conserved. PMID:28420706

The role of steroids in the development of post-partum mental disorders.

PubMed

Paskova, Andrea; Jirak, Roman; Mikesova, Michaela; Adamcova, Karolina; Fartakova, Zdenka; Horakova, Vladimira; Koucky, Michal; Hill, Martin; Hruskovicova, Hana; Starka, Luboslav; Duskova, Michaela; Parizek, Antonin

2014-09-01

Unfavorable post-partum changes to mental well-being affect more than half of all women, and are a risk to the health of both mother and baby. Their effects place strains on health and social systems. Currently, no generally accepted theory exists of the causes and mechanisms of post-partum mental disorders. Literature search up to 2012, using PubMed and search words: neuroactive steroids, post-partum mental disorders, depression, corticotropin-releasing hormone and estrogens. There are several theories for post-partum depression. One is that autoimmune diseases are involved. Others revolve around genes responsible or that lead to increased disposition to the disorder. It is likely however that the process is associated with the separation of the placenta and the fetal zone of fetal adrenal gland, the main sources of corticotropin-releasing hormone and sexual and neuroactive steroids during pregnancy, and the ability of the receptor system to adapt to these changes. The central nervous system is able to produce neurosteroids, but the drop in levels of peripheral steroids likely leads to a sudden deficit in neuroinhibitory steroids modulating ionotropic receptors in the brain. Post-partum depression is a multifactorial disease with unknown etiology. It is probably associated with sudden changes in the production of hormones influencing the nervous system, and on the other hand the ability of the receptor system to adapt to these changes. When the relative changes in concentrations of hormones, rather than their absolute levels, is likely more important.

Modulatory Effects of Sex Steroids Progesterone and Estradiol on Odorant Evoked Responses in Olfactory Receptor Neurons

PubMed Central

Scholz, Paul; Mohrhardt, Julia; Gisselmann, Günter; Hatt, Hanns

2016-01-01

The influence of the sex steroid hormones progesterone and estradiol on physiology and behavior during menstrual cycles and pregnancy is well known. Several studies indicate that olfactory performance changes with cyclically fluctuating steroid hormone levels in females. Knowledge of the exact mechanisms behind how female sex steroids modulate olfactory signaling is limited. A number of different known genomic and non-genomic actions that are mediated by progesterone and estradiol via interactions with different receptors may be responsible for this modulation. Next generation sequencing-based RNA-Seq transcriptome data from the murine olfactory epithelium (OE) and olfactory receptor neurons (ORNs) revealed the expression of several membrane progestin receptors and the estradiol receptor Gpr30. These receptors are known to mediate rapid non-genomic effects through interactions with G proteins. RT-PCR and immunohistochemical staining results provide evidence for progestin and estradiol receptors in the ORNs. These data support the hypothesis that steroid hormones are capable of modulating the odorant-evoked activity of ORNs. Here, we validated this hypothesis through the investigation of steroid hormone effects by submerged electro-olfactogram and whole cell patch-clamp recordings of ORNs. For the first time, we demonstrate that the sex steroid hormones progesterone and estradiol decrease odorant-evoked signals in the OE and ORNs of mice at low nanomolar concentrations. Thus, both of these sex steroids can rapidly modulate the odor responsiveness of ORNs through membrane progestin receptors and the estradiol receptor Gpr30. PMID:27494699

Chemical Interactions of Polyethylene Glycols (PEG) and Glycerol with Protein Functional Groups: Applications to PEG, Glycerol Effects on Protein Processes

PubMed Central

Knowles, DB; Shkel, Irina A; Phan, Noel M; Sternke, Matt; Lingeman, Emily; Cheng, Xian; Cheng, Lixue; O’Connor, Kevin; Record, M. Thomas

2015-01-01

Here we obtain the data needed to predict chemical interactions of polyethylene glycols (PEGs) and glycerol with proteins and related organic compounds, and thereby interpret or predict chemical effects of PEGs on protein processes. To accomplish this we determine interactions of glycerol and tetraEG with >30 model compounds displaying the major C, N, and O functional groups of proteins. Analysis of these data yields coefficients (α-values) quantifying interactions of glycerol, tetraEG and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with aromatic C, amide N, and cationic N, but unfavorably with amide O, carboxylate O and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliphatic C are quite favorable, while interactions of glycerol and PEG end groups with aliphatic C are not. Hence tetraEG and PEG 300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD) while glycerol, di- and mono-ethylene glycol are stabilizers. Favorable interactions with aromatic and aliphatic C explain why PEG400 greatly increases the solubility of aromatic hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α-values reported here, chemical contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA), and separated from excluded volume effects. PMID:25962980

Sex-Dependent Expression of Caveolin 1 in Response to Sex Steroid Hormones Is Closely Associated with Development of Obesity in Rats

PubMed Central

Mukherjee, Rajib; Kim, Sang Woo; Choi, Myung Sook; Yun, Jong Won

2014-01-01

Caveolin-1 (CAV1) is a conserved group of structural membrane proteins that form special cholesterol and sphingolipid-rich compartments, especially in adipocytes. Recently, it has been reported that CAV1 is an important target protein in sex hormone-dependent regulation of various metabolic pathways, particularly in cancer and diabetes. To clarify distinct roles of CAV1 in sex-dependent obesity development, we investigated the effects of high fat diet (HFD) and sex steroid hormones on CAV1 expression in adipose tissues of male and female rats. Results of animal experiments revealed that estrogen (17-β-estradiol, E2) and androgen (dihydrotestosterone, DHT) had opposite effects on body weight gain as well as on the regulation of CAV1, hormone sensitive lipase (HSL) and uncoupling protein 1 (UCP1) in adipose tissues. Furthermore, sex hormone receptors and aromatase were differentially expressed in a sex-dependent manner in response to E2 and DHT treatments. In vivo data were confirmed using 3T3-L1 and HIB1B cell lines, where Cav1 knock down stimulated lipogenesis but suppressed sex hormone receptor signaling proteins. Most importantly, co-immunoprecipitation enabled the identification of previously unrecognized CAV1-interacting mitochondrial or lipid oxidative pathway proteins in adipose tissues. Taken together, current data showed that CAV1 may play important preventive role in the development of obesity, with more prominent effects in females, and proved to be an important target protein for the hormonal regulation of adipose tissue metabolism by manipulating sex hormone receptors and mitochondrial oxidative pathways. Therefore, we can report, for the first time, the molecular mechanism underlying the effects of sex steroid hormones in the sex-dimorphic regulation of CAV1. PMID:24608114

Steroids induce acetylcholine receptors on cultured human muscle: Implications for myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplan, I.; Blakely, B.T.; Pavlath, G.K.

1990-10-01

Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. The authors show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. The results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immunemore » system but also a direct effect on muscle. They propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.« less

Regulation of Adrenocortical Steroid Hormone Production by RhoA-Diaphanous 1 Signaling and the Cytoskeleton

PubMed Central

Sewer, Marion B.; Li, Donghui

2012-01-01

The production of glucocorticoids and aldosterone in the adrenal cortex is regulated at multiple levels. Biosynthesis of these hormones is initiated when cholesterol, the substrate, enters the inner mitochondrial membrane for conversion to pregnenolone. Unlike most metabolic pathways, the biosynthesis of adrenocortical steroid hormones is unique because some of the enzymes are localized in mitochondria and others in the endoplasmic reticulum (ER). Although much is known about the factors that control the transcription and activities of the proteins that are required for steroid hormone production, the parameters that govern the exchange of substrates between the ER and mitochondria are less well understood. This short review summarizes studies that have begun to provide insight into the role of the cytoskeleton, mitochondrial transport, and the physical interaction of the ER and mitochondria in the production of adrenocortical steroid hormones. PMID:23186810

Transsynaptic trophic effects of steroid hormones in an avian model of adult brain plasticity

PubMed Central

Brenowitz, Eliot A.

2014-01-01

The avian song control system provides an excellent model for studying transsynaptic trophic effects of steroid sex hormones. Seasonal changes in systemic testosterone (T) and its metabolites regulate plasticity of this system. Steroids interact with the neurotrophin brain-derived neurotrophic factor (BDNF) to influence cellular processes of plasticity in nucleus HVC of adult birds, including the addition of newborn neurons. This interaction may also occur transsynpatically; T increases the synthesis of BDNF in HVC, and BDNF protein is then released by HVC neurons on to postsynaptic cells in nucleus RA where it has trophic effects on activity and morphology. Androgen action on RA neurons increases their activity and this has a retrograde trophic effect on the addition of new neurons to HVC. The functional linkage of sex steroids to BDNF may be of adaptive value in regulating the trophic effects of the neurotrophin and coordinating circuit function in reproductively relevant contexts. PMID:25285401

The Fate of Synthetic and Endogenous Hormones Used in the US Beef and Dairy Industries and the Potential for Human Exposure.

PubMed

Kolok, Alan S; Ali, Jonathan M; Rogan, Eleanor G; Bartelt-Hunt, Shannon L

2018-05-12

Growth-enhancing chemicals used by the beef and dairy industries may be bioavailable to humans via milk, meat, and other environmental matrices. This review evaluates the potential for environmental transport and bioavailability of the active chemical to humans. Bovine somatostatin is detectable in milk; however, there is no evidence that the protein persists in the environment nor that it is active in humans. In contrast, steroids are transported through milk and meat to humans where they may exert biological activity. Furthermore, environmental matrices such as raw water and dust may also allow for the environmental transport and bioavailability of steroids to humans. Endogenous and exogenous steroids can be found in the meat, milk, and waste materials produced by cattle. While the concentrations may be low, exposure to these matrices, most notably dairy products made with whole milk, can be a source of exogenous steroids to humans.

Steroid Receptor Coactivator-1 Knockdown Decreases Synaptic Plasticity and Impairs Spatial Memory in the Hippocampus of Mice.

PubMed

Bian, Chen; Huang, Yan; Zhu, Haitao; Zhao, Yangang; Zhao, Jikai; Zhang, Jiqiang

2018-05-01

Steroids have been demonstrated to play profound roles in the regulation of hippocampal function by acting on their receptors, which need coactivators for their transcriptional activities. Previous studies have shown that steroid receptor coactivator-1 (SRC-1) is the predominant coactivator in the hippocampus, but its exact role and the underlying mechanisms remain unclear. In this study, we constructed SRC-1 RNA interference (RNAi) lentiviruses, injected them into the hippocampus of male mice, and then examined the changes in the expression of selected synaptic proteins, CA1 synapse density, postsynaptic density (PSD) thickness, and in vivo long-term potentiation (LTP). Spatial learning and memory behavior changes were investigated using the Morris water maze. We then transfected the lentiviruses into cultured hippocampal cells and examined the changes in synaptic protein and phospho-cyclic AMP response element-binding protein (pCREB) expression. The in vivo results showed that SRC-1 knockdown significantly decreased the expression of synaptic proteins and CA1 synapse density as well as PSD thickness; SRC-1 knockdown also significantly impaired in vivo LTP and disrupted spatial learning and memory. The in vitro results showed that while the expression of synaptic proteins was significantly decreased by SRC-1 knockdown, pCREB expression was also significantly decreased. The above results suggest a pivotal role of SRC-1 in the regulation of hippocampal synaptic plasticity and spatial learning and memory, strongly indicating SRC-1 may serve as a novel therapeutic target for hippocampus-dependent memory disorders. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

FOXA1 promotes tumor progression in prostate cancer and represents a novel hallmark of castration-resistant prostate cancer.

PubMed

Gerhardt, Josefine; Montani, Matteo; Wild, Peter; Beer, Marc; Huber, Fabian; Hermanns, Thomas; Müntener, Michael; Kristiansen, Glen

2012-02-01

Forkhead box protein A1 (FOXA1) modulates the transactivation of steroid hormone receptors and thus may influence tumor growth and hormone responsiveness in prostate cancer. We therefore investigated the correlation of FOXA1 expression with clinical parameters, prostate-specific antigen (PSA) relapse-free survival, and hormone receptor expression in a large cohort of prostate cancer patients at different disease stages. FOXA1 expression did not differ significantly between benign glands from the peripheral zone and primary peripheral zone prostate carcinomas. However, FOXA1 was overexpressed in metastases and particularly in castration-resistant cases, but was expressed at lower levels in both normal and neoplastic transitional zone tissues. FOXA1 levels correlated with higher pT stages and Gleason scores, as well as with androgen (AR) and estrogen receptor expression. Moreover, FOXA1 overexpression was associated with faster biochemical disease progression, which was pronounced in patients with low AR levels. Finally, siRNA-based knockdown of FOXA1 induced decreased cell proliferation and migration. Moreover, in vitro tumorigenicity was inducible by ARs only in the presence of FOXA1, substantiating a functional cooperation between FOXA1 and AR. In conclusion, FOXA1 expression is associated with tumor progression, dedifferentiation of prostate cancer cells, and poorer prognosis, as well as with cellular proliferation and migration and with AR signaling. These findings suggest FOXA1 overexpression as a novel mechanism inducing castration resistance in prostate cancer. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Plasma sex steroid binding in Chiroptera.

PubMed

Kwiecinski, G G; Damassa, D A; Gustafson, A W; Armao, M E

1987-04-01

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.

Covalent modification of proteins by ligands of steroid hormone receptors.

PubMed Central

Takahashi, N; Breitman, T R

1992-01-01

Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in HL-60 cells were labeled by 17 beta-[3H]estradiol (E2), [3H]progesterone (Pg), 1 alpha,25-dihydroxy[3H]vitamin D3 [1,25(OH)2D3], [125I]triiodothyronine (T3), [125I]thyroxine (T4), and [3H]prostaglandin E2 (PGE2). All of these hormones, except PGE2, are ligands of the steroid hormone receptor family. Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2. In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg. Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein. These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3. Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands. Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand. Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4. These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases. 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000. However, these proteins had pI values different from those of RI or RII. These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins. Images PMID:1438281

Ocular surface inflammation, and nerve growth factor level in tears in active thyroid-associated ophthalmopathy.

PubMed

Yoon, Jin Sook; Choi, Soo Hyun; Lee, Joon H; Lee, Sung Jun; Lee, Sang Yeul

2010-02-01

To measure tear nerve growth factor (NGF) concentrations in cases of active thyroid-associated ophthalmopathy (TAO) before and after glucocorticoid treatment, and to correlate NGF levels with disease inflammatory activity and thyroid autoantibody concentration. The study involved 20 patients with active TAO and 20 age- and gender-matched controls. Tear break-up time (BUT) was obtained, the Schirmer test was performed, and tear NGF/total protein ratio was measured in control subjects and patients with active TAO before, and 2 and 4 weeks after, steroid treatment. Tear BUT and Schirmer values significantly increased after 2 and 4 weeks of steroid treatment (p < 0.001 and p = 0.004 respectively). Baseline tear NGF/total protein ratio was higher in patients with active TAO than in control subjects, and the ratio significantly decreased after 2 and 4 weeks of steroid treatment (p < 0.001). Tear NGF/total protein ratio did not correlate with inflammatory activity score, exophthalmos value and thyroid binding inhibiting immunoglobulin (TBII) level (p > 0.05). Tear NGF may have a specific role in ocular surface inflammation, which protects against ocular surface damage in patients with active TAO. Anti-inflammatory treatment significantly reduced the level of NGF in tears, increased tear film stability and production, and decreased congestive symptoms.

An In-Silico Investigation of Phytochemicals as Antiviral Agents Against Dengue Fever

PubMed Central

Powers, Chelsea N.; Setzer, William N.

2016-01-01

Abstract: A virtual screening analysis of our library of phytochemical structures with dengue virus protein targets has been carried out using a molecular docking approach. A total of 2194 plant-derived secondary metabolites have been docked. This molecule set comprised of 290 alkaloids (68 indole alkaloids, 153 isoquinoline alkaloids, 5 quinoline alkaloids, 13 piperidine alkaloids, 14 steroidal alkaloids, and 37 miscellaneous alkaloids), 678 terpenoids (47 monoterpenoids, 169 sesquiterpenoids, 265 diterpenoids, 81 steroids, and 96 triterpenoids), 20 aurones, 81 chalcones, 349 flavonoids, 120 isoflavonoids, 74 lignans, 58 stilbenoids, 169 miscellaneous polyphenolic compounds, 100 coumarins, 28 xanthones, 67 quinones, and 160 miscellaneous phytochemicals. Dengue virus protein targets examined included dengue virus protease (NS2B-NS3pro), helicase (NS3 helicase), methyltransferase (MTase), RNA-dependent RNA polymerase (RdRp), and the dengue virus envelope protein. Polyphenolic compounds, flavonoids, chalcones, and other phenolics were the most numerous of the strongly docking ligands for dengue virus protein targets. PMID:27151482

Characterization of SV-40 Tag rats as a model to study prostate cancer

PubMed Central

2009-01-01

Background Prostate cancer is the second most frequently diagnosed cancer in men. Animal models that closely mimic clinical disease in humans are invaluable tools in the fight against prostate cancer. Recently, a Simian Virus-40 T-antigen (SV-40 Tag) targeted probasin promoter rat model was developed. This model, however, has not been extensively characterized; hence we have investigated the ontogeny of prostate cancer and determined the role of sex steroid receptor and insulin-like growth factor-1 (IGF-1) signaling proteins in the novel SV-40 Tag rat. Methods The SV-40 Tag rat was histopathologically characterized for time to tumor development, incidence and multiplicity and in the ventral, dorsal, lateral and anterior lobes of the prostate. Immunoassay techniques were employed to measure cell proliferation, apoptosis, and sex steroid receptor and growth factor signaling-related proteins. Steroid hormone concentrations were measured via coated well enzyme linked immunosorbent assay (ELISA) kits. Results Prostatic intraepithelial neoplasia (PIN) and well-differentiated prostate cancer developed as early as 2 and 10 weeks of age, respectively in the ventral prostate (VP) followed by in the dorsolateral (DLP). At 8 weeks of age, testosterone and dihydrotestosterone (DHT) concentrations in SV-40 Tag rats were increased when compared to non-transgenic rats. High cell proliferation and apoptotic indices were found in VP and DLP of transgenic rats. Furthermore, we observed increased protein expression of androgen receptor, IGF-1, IGF-1 receptor, and extracellular signal-regulated kinases in the prostates of SV-40 Tag rats. Conclusion The rapid development of PIN and prostate cancer in conjunction with the large prostate size makes the SV-40 Tag rat a useful model for studying prostate cancer. This study provides evidence of the role of sex steroid and growth factor proteins in prostate cancer development and defines appropriate windows of opportunity for preclinical trials and aids in the rational design of chemoprevention, intervention, regression, and therapeutic studies using prostate cancer rodent models. PMID:19171036

5β-Reduced steroids and human Δ(4)-3-ketosteroid 5β-reductase (AKR1D1).

PubMed

Chen, Mo; Penning, Trevor M

2014-05-01

5β-Reduced steroids are non-planar steroids that have a 90° bend in their structure to create an A/B cis-ring junction. This novel property is required for bile-acids to act as emulsifiers, but in addition 5β-reduced steroids have remarkable physiology and may act as potent tocolytic agents, endogenous cardiac glycosides, neurosteroids, and can act as ligands for orphan and membrane bound receptors. In humans there is only a single 5β-reductase gene AKR1D1, which encodes Δ(4)-3-ketosteroid-5β-reductase (AKR1D1). This enzyme is a member of the aldo-keto reductase superfamily, but possesses an altered catalytic tetrad, in which Glu120 replaces the conserved His residue. This predominant liver enzyme generates all 5β-dihydrosteroids in the C19-C27 steroid series. Mutations exist in the AKR1D1 gene, which result in loss of protein stability and are causative in bile-acid deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis

PubMed Central

Laurent, Michaël R.; Hammond, Geoffrey L.; Blokland, Marco; Jardí, Ferran; Antonio, Leen; Dubois, Vanessa; Khalil, Rougin; Sterk, Saskia S.; Gielen, Evelien; Decallonne, Brigitte; Carmeliet, Geert; Kaufman, Jean-Marc; Fiers, Tom; Huhtaniemi, Ilpo T.; Vanderschueren, Dirk; Claessens, Frank

2016-01-01

Sex hormone-binding globulin (SHBG) is the high-affinity binding protein for androgens and estrogens. According to the free hormone hypothesis, SHBG modulates the bioactivity of sex steroids by limiting their diffusion into target tissues. Still, the in vivo physiological role of circulating SHBG remains unclear, especially since mice and rats lack circulating SHBG post-natally. To test the free hormone hypothesis in vivo, we examined total and free sex steroid concentrations and bioactivity on target organs in mice expressing a human SHBG transgene. SHBG increased total androgen and estrogen concentrations via hypothalamic-pituitary feedback regulation and prolonged ligand half-life. Despite markedly raised total sex steroid concentrations, free testosterone was unaffected while sex steroid bioactivity on male and female reproductive organs was attenuated. This occurred via a ligand-dependent, genotype-independent mechanism according to in vitro seminal vesicle organ cultures. These results provide compelling support for the determination of free or bioavailable sex steroid concentrations in medicine, and clarify important comparative differences between translational mouse models and human endocrinology. PMID:27748448

Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

PubMed

Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

2009-10-01

Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

Modulatory effects of steroid hormones, oxytocin, arachidonic acid, forskolin and cyclic AMP on the expression of aquaporin 1 and aquaporin 5 in the porcine uterus during placentation.

PubMed

Skowronska, A; Mlotkowska, P; Okrasa, S; Nielsen, S; Skowronski, M T

2016-04-01

Aquaporins (AQPs) are proteins forming trans-membrane channels responsible for water transport. AQP1 and AQP5 are present in structures of the female reproductive system. In the uterus, these AQPs are involved in water movement between the intraluminal, interstitial and capillary compartments and their uterine expression is essential throughout the pregnancy, including its early stages. Thus, the study aimed to assess the influence of P4 (progesterone), E2 (estradiol), OT (oxytocin), AA (arachidonic acid), cAMP and FSK (forskolin) on the AQP1 and AQP5 mRNA and protein expression in the uterine tissue of gilts on Days 30 - 32 of gestation (the placentation period), following short (3 h) and long (24 h) incubations. Steroid hormones influenced the expression of AQP1 and AQP5; E2 up-regulated, but P4 down-regulated mRNAs of these AQPs, whereas the protein level of studied AQPs was increased by both steroids. OT treatment decreased AQP1 (after 24 h), but increased AQP5 (after 3 h) mRNA expression. Treatment with AA significantly reduced the AQP1 expression at the mRNA level, but stimulated at the protein level. The expression of AQP5 mRNA and protein was stimulated by AA. FSK markedly decreased AQP1 mRNA, but increased of AQP5 after 3-h incubation. In turn, cAMP stimulated and inhibited transcription of AQP5 after 3- and 24-h incubations, respectively. Immunohistochemical analysis confirmed the uterine localization of AQP1 in the apical and basal membranes of endothelial cells and AQP5 in the apical membranes of epithelial cells under control condition. Treatments with P4, E2, AA, cAMP or FSK have caused additional appearance of AQP5 labeling in the basolateral membranes of epithelial cells. These results suggest a participation of steroid hormones (P4 and E2), AA derivatives and cAMP in controlling the expression of AQP1 and AQP5 as well as the distribution of AQP5 in the uterine tissue of pregnant gilts during placentation (Days 30 - 32 of gestation).

A Study of the Mechanisms by Which Adrenocorticotropic Hormone Maintains Adrenal Steroidogenic Responsiveness*

PubMed Central

Ney, Robert L.; Dexter, Richard N.; Davis, Warren W.; Garren, Leonard D.

1967-01-01

Following hypophysectomy in the rat, there was a progressive decline in the rate of adrenal protein synthesis in vivo during the ensuing 24-48 hr, and an accompanying decrease in the acute corticosterone secretory response to an intravenous injection of ACTH. There was a similar decrease in the in vitro conversion of Δ5-pregnenolone, progesterone, and deoxycorticosterone to corticosterone. These in vivo and in vitro effects of hypophysectomy could be reversed by the administration of depot ACTH for an additional 7 hr period. However, if cycloheximide, an inhibitor of protein synthesis, was administered concomitantly with the depot ACTH, then the restorative actions of ACTH on the steroid biosynthetic pathway were prevented. These experiments suggest that ACTH maintains not only the general structure of the adrenal cortex, but also the level of the steroid biosynthetic mechanism, through its effects on adrenal protein synthesis. PMID:4294571

Obesity-induced down-regulation of the mitochondrial translocator protein (TSPO) impairs placental steroid production.

PubMed

Lassance, Luciana; Haghiac, Maricela; Minium, Judi; Catalano, Patrick; Hauguel-de Mouzon, Sylvie

2015-01-01

Low concentrations of estradiol and progesterone are hallmarks of adverse pregnancy outcomes as is maternal obesity. During pregnancy, placental cholesterol is the sole source of sex steroids. Cholesterol trafficking is the limiting step in sex steroid biosynthesis and is mainly mediated by the translocator protein (TSPO), present in the mitochondrial outer membrane. The objective of the study was to investigate the effects of maternal obesity in placental sex steroid biosynthesis and TSPO regulation. One hundred forty-four obese (body mass index 30-35 kg/m(2)) and 90 lean (body mass index 19-25 kg/m(2)) pregnant women (OP and LP, respectively) recruited at scheduled term cesarean delivery. Placenta and maternal blood were collected. This study was conducted at MetroHealth Medical Center (Cleveland, Ohio). Maternal metabolic components (fasting glucose, insulin, leptin, estradiol, progesterone, and total cholesterol) and placental weight were measured. Placenta (mitochondria and membranes separated) and cord blood cholesterol values were verified. The expression and regulation of TSPO and mitochondrial function were analyzed. Plasma estradiol and progesterone concentrations were significantly lower (P < .04) in OP as compared with LP women. Maternal and cord plasma cholesterol were not different between groups. Placental citrate synthase activity and mitochondrial DNA, markers of mitochondrial density, were unchanged, but the mitochondrial cholesterol concentrations were 40% lower in the placenta of OP. TSPO gene and protein expressions were decreased 2-fold in the placenta of OP. In vitro trophoblast activation of the innate immune pathways with lipopolysaccharide and long-chain saturated fatty acids reduced TSPO expression by 2- to 3-fold (P < .05). These data indicate that obesity in pregnancy impairs mitochondrial steroidogenic function through the negative regulation of mitochondrial TSPO.

Association between endogenous sex steroid hormones and insulin-like growth factor proteins in US men.

PubMed

Papatheodorou, Stefania I; Rohrmann, Sabine; Lopez, David S; Bradwin, Gary; Joshu, Corinne E; Kanarek, Norma; Nelson, William G; Rifai, Nader; Platz, Elizabeth A; Tsilidis, Konstantinos K

2014-03-01

Sex steroid hormone concentrations and insulin-like growth factor (IGF) proteins have been independently associated with risk of cancer, chronic diseases, and mortality. However, studies that evaluated the inter-relation between the sex hormones and IGF pathways have provided mixed results. We examined the association between endogenous sex hormones and sex hormone-binding globulin (SHBG) with IGF-1 and IGF-binding protein 3 (IGFBP-3) in a population-based sample of US men. Data from 1,135 men aged 20 years or older participating in the third National Health and Nutrition Examination Survey (NHANES III) were analyzed. Weighted linear regression was used to estimate geometric means and 95 % confidence intervals for IGF-1 and IGFBP-3 concentrations by sex steroid hormones and SHBG after adjusting for age, race/ethnicity, body mass index, waist circumference, alcohol consumption, cigarette smoking, physical activity, diabetes, and mutually adjusting for other sex hormones and SHBG. No significant association was observed between sex steroid hormones, SHBG, and IGF-1 concentrations. Total estradiol (% difference in Q5 - Q1 geometric means -9.7 %; P-trend 0.05) and SHBG (% difference -7.3 %; P-trend 0.02) were modestly inversely associated with IGFBP-3. Total testosterone was modestly inversely associated with IGFBP-3 (% difference -6.2 %; P-trend 0.01), but this association disappeared after adjustment for total estradiol and SHBG (% difference 2.6 %; P-trend 0.23). Androstanediol glucuronide was not associated with IGFBP-3. These findings suggest that there may be inter-relationships between circulating total estradiol, SHBG, and IGFBP-3 concentrations. Future research may consider these inter-relationships when evaluating potential joint effects of the sex hormones and IGF pathways.

The extraction of antimicrobials component of andaliman (Zanthoxylum acanthopodium DC.) and its application on catfish (Pangasius sutchi) fillet

NASA Astrophysics Data System (ADS)

Muzafri, A.; Julianti, E.; Rusmarilin, H.

2018-02-01

Andaliman (Zanthoxylum acanthopodium DC.) is a well known wild species in North Sumatera and used for seasoning in Batak’s traditional cuisine. This study was aimed to examine the phytochemical constituents of andaliman fruit extracts after simple macerated in water, methanol, ethyl acetate and hexana using qualitative phytochemical analysis, and to determine its potential antimicrobial activity against Staphylococus aureus, Escherichia coli and Salmonella sp by using agar well difussion method and minimum inhibitory concentration (MIC). Phytochemicals such as alkaloids, flavonoid, glycosides, saponins, tannins, triterpene/steroid and glycoside anthroquinones were detected in the methanol extracts, but steroids and glycisode antraquinones were absent in the ethyl acetate extract. The ethyl acetate extracts showed maximum zone of inhibition and minimum inhibitory concentration against all the experimental microorganisms. The minimum zone of inhibition was determined in hexane extracts showing less antimicrobial activity against all the experimental microorganisms. The MIC of the ethyl acetate extracts was 0,5% w/v for all tested bacteria. Apllication of ethyl acetate extracts of andaliman fruits showed effective for catfish (Pangasius Sutchi) fillet stored in refrigerator (5 °C) for 3 days.

Steroid synthesis by primary human keratinocytes; implications for skin disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hannen, Rosalind F., E-mail: r.f.hannen@qmul.ac.uk; Michael, Anthony E.; Jaulim, Adil

2011-01-07

Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary humanmore » keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra-adrenal cortisol synthesis, which could be a fundamental pathway in skin biology with implications in psoriasis and atopic dermatitis.« less

Disruption of histone modification and CARM1 recruitment by arsenic represses transcription at glucocorticoid receptor-regulated promoters.

PubMed

Barr, Fiona D; Krohmer, Lori J; Hamilton, Joshua W; Sheldon, Lynn A

2009-08-26

Chronic exposure to inorganic arsenic (iAs) found in the environment is one of the most significant and widespread environmental health risks in the U.S. and throughout the world. It is associated with a broad range of health effects from cancer to diabetes as well as reproductive and developmental anomalies. This diversity of diseases can also result from disruption of metabolic and other cellular processes regulated by steroid hormone receptors via aberrant transcriptional regulation. Significantly, exposure to iAs inhibits steroid hormone-mediated gene activation. iAs exposure is associated with disease, but is also used therapeutically to treat specific cancers complicating an understanding of iAs action. Transcriptional activation by steroid hormone receptors is accompanied by changes in histone and non-histone protein post-translational modification (PTM) that result from the enzymatic activity of coactivator and corepressor proteins such as GRIP1 and CARM1. This study addresses how iAs represses steroid receptor-regulated gene transcription. PTMs on histones H3 and H4 at the glucocorticoid receptor (GR)-activated mouse mammary tumor virus (MMTV) promoter were identified by chromatin immunoprecipitation analysis following exposure to steroid hormone+/-iAs. Histone H3K18 and H3R17 amino acid residues had significantly different patterns of PTMs after treatment with iAs. Promoter interaction of the coactivator CARM1 was disrupted, but the interaction of GRIP1, a p160 coactivator through which CARM1 interacts with a promoter, was intact. Over-expression of CARM1 was able to fully restore and GRIP1 partially restored iAs-repressed transcription indicating that these coactivators are functionally associated with iAs-mediated transcriptional repression. Both are essential for robust transcription at steroid hormone regulated genes and both are associated with disease when inappropriately expressed. We postulate that iAs effects on CARM1 and GRIP1 may underlie some of its therapeutic effects and as well be associated with its toxic effects.

Nicolaus Copernicus and the rapid vascular responses to aldosterone.

PubMed

Barton, Matthias; Meyer, Matthias R

2015-08-01

For decades, rapid steroid responses initiated by membrane receptors have been a primary research focus. G protein-coupled estrogen receptor (GPER) is activated by 17β-estradiol and participates in functional crosstalk with other steroid receptors. With reference to the physician and astronomer Nicolaus Copernicus (1473-1543), who used rigorous scientific approaches to shift paradigms and change dogma, we discuss whether GPER can also be considered an aldosterone receptor. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional characterization of CYP52G3 from Aspergillus oryzae and its application for bioconversion and synthesis of hydroxyl flavanone and steroids.

PubMed

Uno, Tomohide; Yanase, Takeshi; Imaishi, Hiromasa

2017-05-01

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Cytochrome P450 (CYP) proteins are ubiquitously distributed in nature and display a broad range of enzymatic activities. A novel CYP52 (CYP52G3) gene was found in A. oryzae. In this study, we report the functional characterization of CYP52G3. The recombinant protein was expressed heterologously in Escherichia coli, and its membrane fraction isolated. CYP52G3 showed activities for 7-ethoxycoumarin and α-naphtoflavone. Furthermore, CYP52G3 hydroxylated flavanone at the 4' and 6 position and metabolized some hydroxyl-flavanones and steroids. Bioconversion experiments indicated that CYP52G3 could convert flavanone and testosterone in a synthetic medium. The conversion rates of flavanone and testosterone at 24 H were 50% and 70%, respectively. These results support that CYP52G3 could prove a useful enzyme for the efficient production of new compounds from flavonoids and steroids. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

GPER (GPR30): A Nongenomic Receptor (GPCR) for Steroid Hormones with Implications for Cardiovascular Disease and Cancer.

PubMed

Feldman, Ross D; Limbird, Lee E

2017-01-06

Although the rapid effects of steroids, such as estrogen and aldosterone, were postulated originally to be nongenomic, it is now appreciated that activation of such signaling pathways via a steroid-acting G protein-coupled receptor, the G protein estrogen receptor (GPER), has important transcription-dependent outcomes in the regulation of cell growth and programmed cell death secondary to GPER-regulated second-messenger pathways. GPER is expressed ubiquitously and has diverse biological effects, including regulation of endocrine, immune, neuronal, and cardiovascular functions. Perhaps the most biologically important consequences of GPER activation are the regulation of cell growth, migration, and apoptotic cell death. These cell growth regulatory effects, important in cancer biology, are also relevant in the regulation of cardiac and vascular hypertrophy and in the response to ischemia. This review provides a summary of relevant findings of the impact of GPER regulation by either estradiol or aldosterone in in vitro model systems and extends those findings to in vivo studies of direct clinical relevance for development of GPER-directed agents for treatment of cancer and cardiovascular diseases associated with cellular proliferation.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

PubMed Central

Zeiner, M; Gehring, U

1995-01-01

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues. Images Fig. 2 Fig. 3 Fig. 4 PMID:8524784

Simultaneous determination of thirteen different steroid hormones using micro UHPLC-MS/MS with on-line SPE system.

PubMed

Márta, Zoltán; Bobály, Balázs; Fekete, Jenő; Magda, Balázs; Imre, Tímea; Mészáros, Katalin Viola; Bálint, Mária; Szabó, Pál Tamás

2018-02-20

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LOQ). Micro UHPLC coupled to sensitive tandem mass spectrometry provides state of the art solution for such analytical problems. Using on-line SPE with column switching on a micro UHPLC-MS/MS system allowed to decrease LOQ without any complex sample preparation protocol. The presented method is capable of reaching satisfactory low LOQ values for analysis of thirteen different steroid molecules from human plasma without the most commonly used off-line SPE or compound derivatization. Steroids were determined by using two simple sample preparation methods, based on lower and higher plasma steroid concentrations. In the first method, higher analyte concentrations were directly determined after protein precipitation with methanol. The organic phase obtained from the precipitation was diluted with water and directly injected into the LC-MS system. In the second method, low steroid levels were determined by concentrating the organic phase after steroid extraction. In this case, analytes were extracted with ethyl acetate and reconstituted in 90/10 water/acetonitrile following evaporation to dryness. This step provided much lower LOQs, outperforming previously published values. The method has been validated and subsequently applied to clinical laboratory measurement. Copyright © 2017 Elsevier B.V. All rights reserved.

Frank Beach Award Winner: Steroids as Neuromodulators of Brain Circuits and Behavior

PubMed Central

Remage-Healey, Luke

2014-01-01

Neurons communicate primarily via action potentials that transmit information on the timescale of milliseconds. Neurons also integrate information via alterations in gene transcription and protein translation that are sustained for hours to days after initiation. Positioned between these two signaling timescales are the minute-by-minute actions of neuromodulators. Over the course of minutes, the classical neuromodulators (such as serotonin, dopamine, octopamine, and norepinephrine) can alter and/or stabilize neural circuit patterning as well as behavioral states. Neuromodulators allow many flexible outputs from neural circuits and can encode information content into the firing state of neural networks. The idea that steroid molecules can operate as genuine behavioral neuromodulators - synthesized by and acting within brain circuits on a minute-by-minute timescale - has gained traction in recent years. Evidence for brain steroid synthesis at synaptic terminals has converged with evidence for the rapid actions of brain-derived steroids on neural circuits and behavior. The general principle emerging from this work is that the production of steroid hormones within brain circuits can alter their functional connectivity and shift sensory representations by enhancing their information coding. Steroids produced in the brain can therefore change the information content of neuronal networks to rapidly modulate sensory experience and sensorimotor functions. PMID:25110187

Evolution of steroids during pregnancy: Maternal, placental and fetal synthesis.

PubMed

Morel, Yves; Roucher, Florence; Plotton, Ingrid; Goursaud, Claire; Tardy, Véronique; Mallet, Delphine

2016-06-01

Progesterone, estrogens, androgens and glucocorticoids are involved in pregnancy from implantation to parturition. Their biosynthesis and their metabolism result from complex pathways involving the fetus, the placenta and the mother. The absence of expression of some steroïdogenic enzymes as CYP17 in placenta and in adrenal fetal zone and the better determination of the onset and variation of others especially HSD3B2 during the pregnancy explain the production of the steroid hormones. Moreover the consequences of some disorders of steroidogenesis (especially aromatase, POR, CYP11A1 and 21-hydroxylase deficiencies) in fetus and mother during the pregnancy have permit to elucidate these complex pathways. This better knowledge of steroid hormones production associated with their dosages in maternal plasma/urine or amniotic fluid using new specific assays as LC-MS MS could facilitate the follow-up of normal and pathological pregnancies. Moreover, these advances should be a basis to evaluate the impact of multiple pathologies of the pregnancy and pharmacologic and xenobiotic consequences on their metabolism. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Superior effect of combination vs. single steroid therapy in keloid disease: a comparative in vitro analysis of glucocorticoids.

PubMed

Syed, Farhatullah; Singh, Subir; Bayat, Ardeshir

2013-01-01

Keloid disease (KD) is a fibroproliferative disorder of unknown etiology. Current use of corticosteroid injection is partially beneficial with 80% recurrence rate. Additionally, the efficacy of different steroids, alone or in combination as opposed to monotherapy, in treating KD remains unclear. Here, we compared the single and combined efficacy of glucocorticoids-dexamethasone (Dex), triamcinolone (TAC), and methylprednisolone (Medrol)-on primary keloid fibroblasts (KFs) (n = 27) and normal skin (n = 19) fibroblasts at cellular, protein, and messenger RNA levels in vitro. Our results demonstrated that cytotoxicity to steroids was dose dependent. Cell spreading, attachment, and proliferation were significantly (p < 0.05) reduced by Medrol and TAC. Migration and invasion properties of KF were inhibited significantly (p < 0.05) by Medrol and TAC compared with Dex. At both protein and messenger RNA levels, keloid-associated fibrotic markers were significantly (p < 0.05) decreased by Medrol and TAC compared with Dex. However, vascular endothelial growth factor expression was significantly (p = 0.01) decreased by Dex compared with TAC and Medrol. Medrol and TAC caused significant (p < 0.04) apoptosis, whereas Dex inhibited the UV-induced apoptosis and up-regulated survivin. Blocking of glucocorticoid receptor by RU486 inhibited cytoprotective property of Dex and apoptotic properties of TAC and Medrol. Double treatment with Dex + TAC and Dex + Medrol significantly (p < 0.05) induced apoptosis. In conclusion, this is the first study to report the efficacy of three well-known steroids on KF and suggest that combination may be superior than using a single steroid in treating KD. © 2012 by the Wound Healing Society.

Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity.

PubMed

Savino, Simone; Ferrandi, Erica Elisa; Forneris, Federico; Rovida, Stefano; Riva, Sergio; Monti, Daniela; Mattevi, Andrea

2016-06-01

Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+) , and the general architecture of the steroid binding site. Comparison with 7α-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the β-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859-865. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Hindbrain lactate regulates preoptic gonadotropin-releasing hormone (GnRH) neuron GnRH-I protein but not AMPK responses to hypoglycemia in the steroid-primed ovariectomized female rat.

PubMed

Shrestha, P K; Briski, K P

2015-07-09

Steroid positive-feedback activation of the gonadotropin-releasing hormone (GnRH)-pituitary luteinizing hormone (LH) neuroendocrine axis propagates the pre ovulatory LH surge, a crucial component of female reproduction. Our work shows that this key event is restrained by inhibitory metabolic input from hindbrain A2 noradrenergic neurons. GnRH neurons express the ultra-sensitive energy sensor adenosine 5'-monophosphate-activated protein kinase (AMPK); here, we investigated the hypothesis that GnRH nerve cell AMPK and peptide neurotransmitter responses to insulin-induced hypoglycemia are controlled by hindbrain lack of the oxidizable glycolytic end-product L-lactate. Data show that hypoglycemic inhibition of LH release in steroid-primed ovariectomized female rats was reversed by coincident caudal hindbrain lactate infusion. Western blot analyses of laser-microdissected A2 neurons demonstrate hypoglycemic augmentation [Fos, estrogen receptor-beta (ER-β), phosphoAMPK (pAMPK)] and inhibition (dopamine-beta-hydroxylase, GLUT3, MCT2) of protein expression in these cells, responses that were normalized by insulin plus lactate treatment. Hypoglycemia diminished rostral preoptic GnRH nerve cell GnRH-I protein and pAMPK content; the former, but not the latter response was reversed by lactate. Results implicate caudal hindbrain lactoprivic signaling in hypoglycemia-induced suppression of the LH surge, demonstrating that lactate repletion of that site reverses decrements in A2 catecholamine biosynthetic enzyme and GnRH neuropeptide precursor protein expression. Lack of effect of lactate on hypoglycemic patterns of GnRH AMPK activity suggests that this sensor is uninvolved in metabolic-inhibition of positive-feedback-stimulated hypophysiotropic signaling to pituitary gonadotropes. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Tadalafil modulates aromatase activity and androgen receptor expression in a human osteoblastic cell in vitro model.

PubMed

Aversa, A; Fittipaldi, S; Bimonte, V M; Wannenes, F; Papa, V; Francomano, D; Greco, E A; Lenzi, A; Migliaccio, S

2016-02-01

Phosphodiesterase type-5 inhibitor (PDE5i) tadalafil administration in men with erectile dysfunction is associated with increased testosterone/estradiol ratio, leading to hypothesize a potential increased effect of androgen action on target tissues. We aimed to characterize, in a cellular model system in vitro, the potential modulation of aromatase and sex steroid hormone receptors upon exposure to tadalafil (TAD). Human osteoblast-like cells SAOS-2 were chosen as an in vitro model system since osteoblasts are target of steroid hormones. Cells were tested for viability upon TAD exposure, which increased cell proliferation. Then, cells were treated with/without TAD for several times to evaluate potential modulation in PDE5, aromatase (ARO), androgen (AR) and estrogen (ER) receptor expression. Osteoblasts express significant levels of both PDE5 mRNA and protein. Exposure of cells to increasing concentrations of TAD (10(-8)-10(-7) M) decreased PDE5 mRNA and protein expression. Also, TAD inhibited ARO mRNA and protein expression leading to an increase in testosterone levels in the supernatants. Interestingly, TAD increased total AR mRNA and protein expression and decreased ERα, with an increased ratio of AR/ER, suggesting preferential androgenic vs estrogenic pathway activation. Our results demonstrate for the first time that TAD decreases ARO expression and increases AR protein expression in human SAOS-2, strongly suggesting a new control of steroid hormones pathway by PDE5i. These findings might represent the first evidence of translational actions of PDE5i on AR, which leads to hypothesize a growing relevance of this molecule in men with prostate cancer long-term treated with TAD for sexual rehabilitation.

Steroid concentrations in antepartum and postpartum saliva: normative values in women and correlations with serum

PubMed Central

2013-01-01

Background Saliva has been advocated as an alternative to serum or plasma for steroid monitoring. Little normative information is available concerning expected concentrations of the major reproductive steroids in saliva during pregnancy and the extended postpartum. Methods Matched serum and saliva specimens controlled for time of day and collected less than 30 minutes apart were obtained in 28 women with normal singleton pregnancies between 32 and 38 weeks of gestation and in 43 women during the first six months postpartum. Concentrations of six steroids (estriol, estradiol, progesterone, testosterone, cortisol, dehydroepiandrosterone) were quantified in saliva by enzyme immunoassay. Results For most of the steroids examined, concentrations in antepartum saliva showed linear increases near end of gestation, suggesting an increase in the bioavailable hormone component. Observed concentrations were in agreement with the limited data available from previous reports. Modal concentrations of the ovarian steroids were undetectable in postpartum saliva and, when detectable in individual women, approximated early follicular phase values. Only low to moderate correlations between the serum and salivary concentrations were found, suggesting that during the peripartum period saliva provides information that is not redundant to serum. Conclusions Low correlations in the late antepartum may be due to differential rates of change in the total and bioavailable fractions of the circulating steroid in the final weeks of the third trimester as a consequence of dynamic changes in carrier proteins such as corticosteroid binding globulin. PMID:23575245

Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demura, T.; Driscoll, W.J.; Lee, Y.C.

1991-01-01

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinctmore » from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.« less

General overview on structure prediction of twilight-zone proteins.

PubMed

Khor, Bee Yin; Tye, Gee Jun; Lim, Theam Soon; Choong, Yee Siew

2015-09-04

Protein structure prediction from amino acid sequence has been one of the most challenging aspects in computational structural biology despite significant progress in recent years showed by critical assessment of protein structure prediction (CASP) experiments. When experimentally determined structures are unavailable, the predictive structures may serve as starting points to study a protein. If the target protein consists of homologous region, high-resolution (typically <1.5 Å) model can be built via comparative modelling. However, when confronted with low sequence similarity of the target protein (also known as twilight-zone protein, sequence identity with available templates is less than 30%), the protein structure prediction has to be initiated from scratch. Traditionally, twilight-zone proteins can be predicted via threading or ab initio method. Based on the current trend, combination of different methods brings an improved success in the prediction of twilight-zone proteins. In this mini review, the methods, progresses and challenges for the prediction of twilight-zone proteins were discussed.

Predictive factors of clinical response in steroid-refractory ulcerative colitis treated with granulocyte-monocyte apheresis

PubMed Central

D'Ovidio, Valeria; Meo, Donatella; Viscido, Angelo; Bresci, Giampaolo; Vernia, Piero; Caprilli, Renzo

2011-01-01

AIM: To identify factors predicting the clinical response of ulcerative colitis patients to granulocyte-monocyte apheresis (GMA). METHODS: Sixty-nine ulcerative colitis patients (39 F, 30 M) dependent upon/refractory to steroids were treated with GMA. Steroid dependency, clinical activity index (CAI), C reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), values at baseline, use of immunosuppressant, duration of disease, and age and extent of disease were considered for statistical analysis as predictive factors of clinical response. Univariate and multivariate logistic regression models were used. RESULTS: In the univariate analysis, CAI (P = 0.039) and ESR (P = 0.017) levels at baseline were singled out as predictive of clinical remission. In the multivariate analysis steroid dependency [Odds ratio (OR) = 0.390, 95% Confidence interval (CI): 0.176-0.865, Wald 5.361, P = 0.0160] and low CAI levels at baseline (4 < CAI < 7) (OR = 0.770, 95% CI: 0.425-1.394, Wald 3.747, P = 0.028) proved to be effective as factors predicting clinical response. CONCLUSION: GMA may be a valid therapeutic option for steroid-dependent ulcerative colitis patients with mild-moderate disease and its clinical efficacy seems to persist for 12 mo. PMID:21528055

Food protein induced enterocolitis syndrome caused by rice beverage.

PubMed

Caminiti, Lucia; Salzano, Giuseppina; Crisafulli, Giuseppe; Porcaro, Federica; Pajno, Giovanni Battista

2013-05-14

Food protein-induced enterocolitis syndrome (FPIES) is an uncommon and potentially severe non IgE-mediated gastrointestinal food allergy. It is usually caused by cow's milk or soy proteins, but may also be triggered by ingestion of solid foods. The diagnosis is made on the basis of clinical history and symptoms. Management of acute phase requires fluid resuscitation and intravenous steroids administration, but avoidance of offending foods is the only effective therapeutic option.Infant with FPIES presented to our emergency department with vomiting, watery stools, hypothension and metabolic acidosis after ingestion of rice beverage. Intravenous fluids and steroids were administered with good clinical response. Subsequently, a double blind placebo control food challenge (DBPCFC) was performed using rice beverage and hydrolyzed formula (eHF) as placebo. The "rice based formula" induced emesis, diarrhoea and lethargy. Laboratory investigations reveal an increase of absolute count of neutrophils and the presence of faecal eosinophils. The patient was treated with both intravenous hydration and steroids. According to Powell criteria, oral food challenge was considered positive and diagnosis of FPIES induced by rice beverage was made. Patient was discharged at home with the indication to avoid rice and any rice beverage as well as to reintroduce hydrolyzed formula. A case of FPIES induced by rice beverage has never been reported. The present case clearly shows that also beverage containing rice proteins can be responsible of FPIES. For this reason, the use of rice beverage as cow's milk substitute for the treatment of non IgE-mediated food allergy should be avoided.

Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice.

PubMed

Nishimune, Hiroshi; Badawi, Yomna; Mori, Shuuichi; Shigemoto, Kazuhiro

2016-06-20

Presynaptic active zones play a pivotal role as synaptic vesicle release sites for synaptic transmission, but the molecular architecture of active zones in mammalian neuromuscular junctions (NMJs) at sub-diffraction limited resolution remains unknown. Bassoon and Piccolo are active zone specific cytosolic proteins essential for active zone assembly in NMJs, ribbon synapses, and brain synapses. These proteins are thought to colocalize and share some functions at active zones. Here, we report an unexpected finding of non-overlapping localization of these two proteins in mouse NMJs revealed using dual-color stimulated emission depletion (STED) super resolution microscopy. Piccolo puncta sandwiched Bassoon puncta and aligned in a Piccolo-Bassoon-Piccolo structure in adult NMJs. P/Q-type voltage-gated calcium channel (VGCC) puncta colocalized with Bassoon puncta. The P/Q-type VGCC and Bassoon protein levels decreased significantly in NMJs from aged mouse. In contrast, the Piccolo levels in NMJs from aged mice were comparable to levels in adult mice. This study revealed the molecular architecture of active zones in mouse NMJs at sub-diffraction limited resolution, and described the selective degeneration mechanism of active zone proteins in NMJs from aged mice. Interestingly, the localization pattern of active zone proteins described herein is similar to active zone structures described using electron microscope tomography.

Steroid hydroxylation by basidiomycete peroxygenases: a combined experimental and computational study.

PubMed

Babot, Esteban D; Del Río, José C; Cañellas, Marina; Sancho, Ferran; Lucas, Fátima; Guallar, Víctor; Kalum, Lisbeth; Lund, Henrik; Gröbe, Glenn; Scheibner, Katrin; Ullrich, René; Hofrichter, Martin; Martínez, Angel T; Gutiérrez, Ana

2015-06-15

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106-118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Steroid Hydroxylation by Basidiomycete Peroxygenases: a Combined Experimental and Computational Study

PubMed Central

Babot, Esteban D.; del Río, José C.; Cañellas, Marina; Sancho, Ferran; Lucas, Fátima; Guallar, Víctor; Kalum, Lisbeth; Lund, Henrik; Gröbe, Glenn; Scheibner, Katrin; Ullrich, René; Hofrichter, Martin; Martínez, Angel T.

2015-01-01

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106–118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed. PMID:25862224

Mechanisms of oocyte development in European sea bass (Dicentrarchus labrax L.): investigations via application of unilateral ovariectomy.

PubMed

García-López, Ángel; Sánchez-Amaya, María I; Tyler, Charles R; Prat, Francisco

2011-08-01

Unilateral ovariectomy (ULO) was performed in European sea bass (Dicentrarchus labrax L.) during late pre-vitellogenesis/early vitellogenesis. Plasma steroid levels and the expression of a suite of potential oogenesis-relevant genes in the ovary, brain, and pituitary were evaluated with the aim of understanding their involvement in the compensatory oocyte development occurring within the remaining ovarian lobe. After 69 days of surgery the remaining ovarian lobe in ULO fish was gravimetrically equivalent to an intact-paired ovary of sham operated, control fish. This compensatory ovarian growth was based on an increased number of early perinucleolar oocytes and mid-late stage vitellogenic follicles without an apparent recruitment of primary oocytes into the secondary growth phase. Plasma steroid levels were similar in ULO and control females at all time points analyzed, suggesting an increased steroid production of the remaining ovarian lobe in hemi-castrated females. Results of the gene expression survey conducted indicate that the signaling pathways mediated by Fsh and Gnrh1 constitute the central axes orchestrating the observed ovarian compensatory growth. In addition, steroid receptors, Star protein, Igfs, and members of the transforming growth factor beta superfamily including anti-Mullerian hormone and bone morphogenetic protein 4 were identified as potentially relevant players within this process, although their specific actions and interactions remain to be established. Our results demonstrate that ULO provides an excellent in vivo model for elucidating the interconnected endocrine and molecular mechanisms controlling oocyte development in European sea bass.

NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone

PubMed Central

Awata, Junya; Takada, Saeko; Standley, Clive; Lechtreck, Karl F.; Bellvé, Karl D.; Pazour, Gregory J.; Fogarty, Kevin E.; Witman, George B.

2014-01-01

ABSTRACT The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about the function of NPHP4, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein. Therefore, these two proteins, which are incorporated into the transition zone independently of each other, define different domains of the transition zone. An nphp4-null mutant forms flagella with nearly normal length, ultrastructure and intraflagellar transport. When fractions from isolated wild-type and nphp4 flagella were compared, few differences were observed between the axonemes, but the amounts of certain membrane proteins were greatly reduced in the mutant flagella, and cellular housekeeping proteins >50 kDa were no longer excluded from mutant flagella. Therefore, NPHP4 functions at the transition zone as an essential part of a barrier that regulates both membrane and soluble protein composition of flagella. The phenotypic consequences of NPHP4 mutations in humans likely follow from protein mislocalization due to defects in the transition zone barrier. PMID:25150219

Use of estetrol with other steroids for attenuation of neonatal hypoxic-ischemic brain injury: to combine or not to combine?

PubMed

Tskitishvili, Ekaterine; Pequeux, Christel; Munaut, Carine; Viellevoye, Renaud; Nisolle, Michelle; Noël, Agnes; Foidart, Jean-Michel

2016-06-07

Estetrol (E4), estradiol (E2) and progesterone (P4) have important antioxidative and neuroprotective effects in neuronal system. We aimed to study the consequence of combined steroid therapy in neonatal hypoxic-ischemic encephalopathy (HIE). In vitro the effect of E4 combined with other steroids on oxidative stress and the cell viability in primary hippocampal cultures was evaluated by lactate dehydrogenase and cell survival assays. In vivo neuroprotective and therapeutic efficacy of E4 combined with other steroids was studied in HIE model of immature rats. The rat pups rectal temperature, body and brain weights were evaluated.The hippocampus and the cortex were investigated by histo/immunohistochemistry: intact cell number counting, expressions of markers for early gray matter lose, neuro- and angiogenesis were studied. Glial fibrillary acidic protein was evaluated by ELISA in blood samples. In vitro E4 and combinations of high doses of E4 with P4 and/or E2 significantly diminished the LDH activity and upregulated the cell survival.In vivopretreatment or treatment by different combinations of E4 with other steroids had unalike effects on body and brain weight, neuro- and angiogenesis, and GFAP expression in blood. The combined use of E4 with other steroids has no benefit over the single use of E4.

Placental sulfatase deficiency: maternal and fetal expression of steroid sulfatase deficiency and X-linked ichthyosis.

PubMed

Bradshaw, K D; Carr, B R

1986-07-01

PSD-X-linked ichthyosis are manifestations of a similar disorder of an inborn error of metabolism characterized by a deficiency of steroid sulfatase. The decreased enzyme activity is due to the absence of the expression of enzyme (steroid sulfatase) protein. Affected individuals with this disorder are males (X-linked inheritance) with a frequency of 1/2000 to 1/6000 births. Homozygous females from cosanguineous marriages have been reported with this disorder. The diagnosis is suspected and confirmed by: Low estriol excretion; Negative DHEAS loading test Increased DHEAS in amnionic fluid; Normal DHEAS in cord plasma; Possible delayed or abnormal labor patterns; Decreased sulfatase activity in the placenta, fibroblast, erythrocytes, lymphocytes or leukocytes of affected individuals; Development of ichthyosis in male infants at 2 to 3 months of age.

9,11-Secosteroids and polyhydroxylated steroids from two South China Sea soft corals Sarcophyton trocheliophorum and Sinularia flexibilis.

PubMed

Chen, Wen-Ting; Liu, Hai-Li; Yao, Li-Gong; Guo, Yue-Wei

2014-12-01

A new 9,11-secosteroid, 25(26)-dehydrosarcomilasterol (1), two new polyhydroxylated steroids, 7α-hydroxy-crassarosterol A (2) and 11-acetoxy-7α-hydroxy-crassarosterol A (3), together with three known related ones (4-6), were isolated from the South China Sea soft corals Sarcophyton trocheliophorum and Sinularia flexibilis, respectively. The structures of the new steroids were elucidated on the basis of extensive spectroscopic analyses, comparison with the literature data and chemical correlation. Compound 2 exhibited a moderate protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with an IC50 value of 33.05μM. Compounds 1-3 showed weak in vitro cytotoxicities against the tumor cell lines K562 and HL-60. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes

PubMed Central

Sinars, Cindy R.; Cheung-Flynn, Joyce; Rimerman, Ronald A.; Scammell, Jonathan G.; Smith, David F.; Clardy, Jon

2003-01-01

The ability to bind immunosuppressive drugs such as cyclosporin and FK506 defines the immunophilin family of proteins, and the FK506-binding proteins form the FKBP subfamily of immunophilins. Some FKBPs, notably FKBP12 (the 12-kDa FK506-binding protein), have defined roles in regulating ion channels or cell signaling, and well established structures. Other FKBPs, especially the larger ones, participate in important biological processes, but their exact roles and the structural bases for these roles are poorly defined. FKBP51 (the 51-kDa FKBP) associates with heat shock protein 90 (Hsp90) and appears in functionally mature steroid receptor complexes. In New World monkeys, FKBP51 has been implicated in cortisol resistance. We report here the x-ray structures of human FKBP51, to 2.7 Å, and squirrel monkey FKBP51, to 2.8 Å, by using multiwavelength anomalous dispersion phasing. FKBP51 is composed of three domains: two consecutive FKBP domains and a three-unit repeat of the TPR (tetratricopeptide repeat) domain. This structure of a multi-FKBP domain protein clarifies the arrangement of these domains and their possible interactions with other proteins. The two FKBP domains differ by an insertion in the second that affects the formation of the progesterone receptor complex. PMID:12538866

Paxillin and embryonic PolyAdenylation Binding Protein (ePABP) engage to regulate androgen-dependent Xenopus laevis oocyte maturation - A model of kinase-dependent regulation of protein expression.

PubMed

Miedlich, Susanne U; Taya, Manisha; Young, Melissa Rasar; Hammes, Stephen R

2017-06-15

Steroid-triggered Xenopus laevis oocyte maturation is an elegant physiologic model of nongenomic steroid signaling, as it proceeds completely independent of transcription. We previously demonstrated that androgens are the main physiologic stimulator of oocyte maturation in Xenopus oocytes, and that the adaptor protein paxillin plays a crucial role in mediating this process through a positive feedback loop in which paxillin first enhances Mos protein translation, ensued by Erk2 activation and Erk-dependent phosphorylation of paxillin on serine residues. Phosphoserine-paxillin then further augments Mos protein translation and downstream Erk2 activation, resulting in meiotic progression. We hypothesized that paxillin enhances Mos translation by interacting with embryonic PolyAdenylation Binding Protein (ePABP) on polyadenylated Mos mRNA. Knockdown of ePABP phenocopied paxillin knockdown, with reduced Mos protein expression, Erk2 and Cdk1 activation, as well as oocyte maturation. In both Xenopus oocytes and mammalian cells (HEK-293), paxillin and ePABP constitutively interacted. Testosterone (Xenopus) or EGF (HEK-293) augmented ePABP-paxillin binding, as well as ePABP binding to Mos mRNA (Xenopus), in an Erk-dependent fashion. Thus, ePABP and paxillin work together in an Erk-dependent fashion to enhance Mos protein translation and promote oocyte maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation.

PubMed

Grishkovskaya, Irina; Avvakumov, George V; Hammond, Geoffrey L; Catalano, Maria G; Muller, Yves A

2002-08-30

The amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG) contains a single high affinity steroid-binding site. Crystal structures of this domain in complex with several different steroid ligands have revealed that estradiol occupies the SHBG steroid-binding site in an opposite orientation when compared with 5 alpha-dihydrotestosterone or C19 androgen metabolites (5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 beta,17 alpha-diol) or the synthetic progestin levonorgestrel. Substitution of specific residues within the SHBG steroid-binding site confirmed that Ser(42) plays a key role in determining high affinity interactions by hydrogen bonding to functional groups at C3 of the androstanediols and levonorgestrel and the hydroxyl at C17 of estradiol. Among residues participating in the hydrogen bond network with hydroxy groups at C17 of C19 steroids or C3 of estradiol, Asp(65) appears to be the most important. The different binding mode of estradiol is associated with a difference in the position/orientation of residues (Leu(131) and Lys(134)) in the loop segment (Leu(131)-His(136)) that covers the steroid-binding site as well as others (Leu(171)-Lys(173) and Trp(84)) on the surface of human SHBG and may provide a basis for ligand-dependent interactions between SHBG and other macromolecules. These new crystal structures have also enabled us to construct a simple space-filling model that can be used to predict the characteristics of novel SHBG ligands.

High pre-transplant serum nitrate levels predict risk of acute steroid-refractory graft-versus-host disease in the absence of statin therapy

PubMed Central

Dietrich, Sascha; Okun, Jürgen G.; Schmidt, Kathrin; Falk, Christine S.; Wagner, Andreas H.; Karamustafa, Suzan; Radujkovic, Aleksandar; Hegenbart, Ute; Ho, Anthony D.; Dreger, Peter; Luft, Thomas

2014-01-01

Steroid-refractory graft-versus-host disease is a life-threatening complication after allogeneic stem cell transplantation. Evidence is accumulating that steroid-refractory graft-versus-host disease is associated with endothelial distress. Endothelial cell homeostasis is regulated by nitric oxide, and serum nitrates are derived from nitric oxide synthase activity or dietary sources. In this retrospective study based on 417 patients allografted at our institution we investigated whether quantification of serum nitrates could predict steroid-refractory graft-versus-host disease. Elevated pre-transplant levels of serum nitrates (>26.5 μM) predicted steroid-refractory graft-versus-host disease (P=0.026) and non-relapse mortality (P=0.028), particularly in combination with high pre-transplant angiopoietin-2 levels (P=0.0007 and P=0.021, respectively). Multivariate analyses confirmed serum nitrates as independent predictors of steroid-refractory graft-versus-host disease and non-relapse mortality. Differences in serum nitrate levels did not correlate with serum levels of tumor necrosis factor or C-reactive protein or expression of inducible nitric oxide synthase in blood cells. Patients with high pre-transplant nitrate levels had significantly reduced rates of refractory graft-versus-host disease (P=0.031) when pravastatin was taken. In summary, patients at high risk of developing steroid-refractory graft-versus-host disease could be identified prior to transplantation by serum markers linked to endothelial cell function. Retrospectively, statin medication was associated with a reduced incidence of refractory graft-versus-host disease in this endothelial high-risk cohort. PMID:24142995

Liprin-α3 controls vesicle docking and exocytosis at the active zone of hippocampal synapses.

PubMed

Wong, Man Yan; Liu, Changliang; Wang, Shan Shan H; Roquas, Aram C F; Fowler, Stephen C; Kaeser, Pascal S

2018-02-27

The presynaptic active zone provides sites for vesicle docking and release at central nervous synapses and is essential for speed and accuracy of synaptic transmission. Liprin-α binds to several active zone proteins, and loss-of-function studies in invertebrates established important roles for Liprin-α in neurodevelopment and active zone assembly. However, Liprin-α localization and functions in vertebrates have remained unclear. We used stimulated emission depletion superresolution microscopy to systematically determine the localization of Liprin-α2 and Liprin-α3, the two predominant Liprin-α proteins in the vertebrate brain, relative to other active-zone proteins. Both proteins were widely distributed in hippocampal nerve terminals, and Liprin-α3, but not Liprin-α2, had a prominent component that colocalized with the active-zone proteins Bassoon, RIM, Munc13, RIM-BP, and ELKS. To assess Liprin-α3 functions, we generated Liprin-α3-KO mice by using CRISPR/Cas9 gene editing. We found reduced synaptic vesicle tethering and docking in hippocampal neurons of Liprin-α3-KO mice, and synaptic vesicle exocytosis was impaired. Liprin-α3 KO also led to mild alterations in active zone structure, accompanied by translocation of Liprin-α2 to active zones. These findings establish important roles for Liprin-α3 in active-zone assembly and function, and suggest that interplay between various Liprin-α proteins controls their active-zone localization.

[Treatment option for intralamellar corneo-scleral abscesses in the zone of tunnel incision after phacoemulsification].

PubMed

Kasparova, E A; Kasparov, A A

2012-01-01

The article provides data on clinical diagnosis (4 patients, 4 eyes) and early surgical treatment of corneal intralamellar abscesses (ICA) in the zone of corneo-scleral tunnel incision after phacoemulsification (PE). ICA developed via corneo-scleral incision was a relatively reliable biomicroscopic sign of the beginning chronic endophthalmitis. Despite of massive nonsurgical treatment (antibiotics, glucocorticoids, non-steroid anti-inflammatory agents etc.) inflammation was not arrested completely, it recurred within 2,5-6 months after PE. Reconstructive conjunctivo-sclerokeratoplasty resulted in arrest of inflammation and visual function recovery in 3 patients. In one patient this procedure was not possible to perform because of total loss of visual functions due to chronic endophthalmitis.

Protein Interaction Analysis Provides a Map of the Spatial and Temporal Organization of the Ciliary Gating Zone.

PubMed

Takao, Daisuke; Wang, Liang; Boss, Allison; Verhey, Kristen J

2017-08-07

The motility and signaling functions of the primary cilium require a unique protein and lipid composition that is determined by gating mechanisms localized at the base of the cilium. Several protein complexes localize to the gating zone and may regulate ciliary protein composition; however, the mechanisms of ciliary gating and the dynamics of the gating components are largely unknown. Here, we used the BiFC (bimolecular fluorescence complementation) assay and report for the first time on the protein-protein interactions that occur between ciliary gating components and transiting cargoes during ciliary entry. We find that the nucleoporin Nup62 and the C termini of the nephronophthisis (NPHP) proteins NPHP4 and NPHP5 interact with the axoneme-associated kinesin-2 motor KIF17 and thus spatially map to the inner region of the ciliary gating zone. Nup62 and NPHP4 exhibit rapid turnover at the transition zone and thus define dynamic components of the gate. We find that B9D1, AHI1, and the N termini of NPHP4 and NPHP5 interact with the transmembrane protein SSTR3 and thus spatially map to the outer region of the ciliary gating zone. B9D1, AHI1, and NPHP5 exhibit little to no turnover at the transition zone and thus define components of a stable gating structure. These data provide the first comprehensive map of the molecular orientations of gating zone components along the inner-to-outer axis of the ciliary gating zone. These results advance our understanding of the functional roles of gating zone components in regulating ciliary protein composition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Unc-51 controls active zone density and protein composition by downregulating ERK signaling.

PubMed

Wairkar, Yogesh P; Toda, Hirofumi; Mochizuki, Hiroaki; Furukubo-Tokunaga, Katsuo; Tomoda, Toshifumi; Diantonio, Aaron

2009-01-14

Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. Here we use a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability.

Triiodothyronine stimulates VEGF expression and secretion via steroids and HIF-1α in murine Leydig cells.

PubMed

Dhole, Bodhana; Gupta, Surabhi; Venugopal, Senthil Kumar; Kumar, Anand

2018-06-01

Leydig cells are the principal steroidogenic cells of the testis. Leydig cells also secrete a number of growth factors including vascular endothelial growth factor (VEGF) which has been shown to regulate both testicular steroidogenesis and spermatogenesis. The thyroid hormone, T 3, is known to stimulate steroidogenesis in Leydig cells. T 3 has also been shown to stimulate VEGF production in a variety of cell lines. However, studies regarding the effect of T 3 on VEGF synthesis and secretion by the Leydig cells were lacking. Therefore, we investigated the effect of T 3 on VEGF synthesis and secretion in a mouse Leydig tumour cell line, MLTC-1. The effect of T 3 was compared with that of LH/cAMP and hypoxia, two known stimulators of Leydig cell functions. The cells were treated with T 3 , 8-Br-cAMP (a cAMP analogue), or CoCl 2 (a hypoxia mimetic) and VEGF secreted in the cell supernatant was measured using ELISA. The mRNA levels of VEGF were measured by quantitative RT-PCR. In the MLTC-1 cells, T 3 , 8-Br-cAMP, and CoCl 2 stimulated VEGF mRNA levels and the protein secretion. T 3 also increased steroid secretion as well as HIF-1α protein levels, two well-established upstream regulators of VEGF. Inhibitors of steroidogenesis as well as HIF-1α resulted in inhibition of T 3 -stimulated VEGF secretion by the MLTC-1 cells. This suggested a mediatory role of steroids and HIF-1α protein in T 3 -stimulated VEGF secretion by MLTC-1 cells. The mediation by steroids and HIF-1α were independent of each other. 8-Br-cAMP: 8-bromo - 3', 5' cyclic adenosine monophosphate; CoCl 2 : cobalt chloride; HIF-1α: hypoxia inducible factor -1α; LH: luteinizing hormone; T 3 : 3, 5, 3'-L-triiodothyronine; VEGF: vascular endothelial growth factor.

Making muscles "stronger": exercise, nutrition, drugs.

PubMed

Aagaard, P

2004-06-01

As described in this review, maximal muscle strength is strongly influenced by resistive-types of exercise, which induce adaptive changes in both neuromuscular function and muscle morphology. Further, timed intake of protein in conjunction with resistance training elicit greater strength and muscle size gains than resistance training alone. Creatine supplementation amplifies the hypertrophic response to resistance training, although some individuals may not respond positively. Locally produced muscle growth factors are upregulated during creatine supplementation, which contributes to increase the responsiveness of muscle cells to intensive training stimuli. Usage of anabolic steroids boosts muscle hypertrophy beyond inherent genetical limits, not only by increasing the DNA transcription rate for myofibrillar proteins but also by increasing the nucleus-to-cytoplasm ratio due to accelerated activation of myogenic satellite cells. However, severe tissue damaging effects exist with anabolic steroids, some of which are irreversible.

Steroid receptor coactivator-1 mediates letrozole induced downregulation of postsynaptic protein PSD-95 in the hippocampus of adult female rats.

PubMed

Liu, Mengying; Huangfu, Xuhong; Zhao, Yangang; Zhang, Dongmei; Zhang, Jiqiang

2015-11-01

Hippocampus local estrogen which is converted from androgen that catalyzed by aromatase has been shown to play important roles in the regulation of learning and memory as well as cognition through action on synaptic plasticity, but the underlying mechanisms are poorly understood. Steroid receptor coactivator-1 (SRC-1) is one of the coactivators of steroid nuclear receptors; it is widely distributed in brain areas that related to learning and memory, reproductive regulation, sensory and motor information integration. Previous studies have revealed high levels of SRC-1 immunoreactivities in the hippocampus; it is closely related to the levels of synaptic proteins such as PSD-95 under normal development or gonadectomy, but its exact roles in the regulation of these proteins remains unclear. In this study, we used aromatase inhibitor letrozole in vivo and SRC-1 RNA interference in vitro to investigate whether SRC-1 mediated endogenous estrogen regulation of hippocampal PSD-95. The results revealed that letrozole injection synchronously decreased hippocampal SRC-1 and PSD-95 in a dose-dependant manner. Furthermore, when SRC-1 specific shRNA pool was applied to block the expression of SRC-1 in the primary hippocampal neuron culture, both immunocytochemistry and Western blot revealed that levels of PSD-95 were also decreased significantly. Taking together, these results provided the first evidence that SRC-1 mediated endogenous estrogen regulation of hippocampal synaptic plasticity by targeting the expression of synaptic protein PSD-95. Additionally, since letrozole is frequently used to treat estrogen-sensitive breast cancer, the above results also indicate its potential side effects in clinical administration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcriptional regulation of genes related to progesterone production.

PubMed

Mizutani, Tetsuya; Ishikane, Shin; Kawabe, Shinya; Umezawa, Akihiro; Miyamoto, Kaoru

2015-01-01

Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3β-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

Ketoconazole inhibition of testicular secretion of testosterone and displacement of steroid hormones from serum transport proteins.

PubMed Central

Grosso, D S; Boyden, T W; Pamenter, R W; Johnson, D G; Stevens, D A; Galgiani, J N

1983-01-01

In vivo perfusion of canine testes with ketoconazole inhibited the stimulation of testosterone production by human chorionic gonadotropin in a dose-dependent manner. Ketoconazole also selectively displaced steroids from serum-binding globulins. Dihydrotestosterone and estradiol binding to sex hormone-binding globulin were inhibited by ketoconazole. Cortisol binding to corticosteroid-binding globulin was unaffected. The concentrations of ketoconazole that inhibited human chorionic gonadotropin stimulation of testicular androgen production and displaced sex steroids from sex hormone-binding globulin were in the range of blood levels found in patients on higher therapeutic dosage regimens. Suppression of testicular testosterone synthesis and displacement of estrogens from sex hormone-binding globulin may decrease the androgen/estrogen ratio of the blood and contribute to the development of gynecomastia that has been reported in some ketoconazole-treated patients. PMID:6301363

Identification and characterization of the steroid 15α-hydroxylase gene from Penicillium raistrickii.

PubMed

Jia, Longgang; Dong, Jianzhang; Wang, Ruijie; Mao, Shuhong; Lu, Fuping; Singh, Suren; Wang, Zhengxiang; Liu, Xiaoguang

2017-08-01

Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions.

Stimulation of progesterone production by phorbol-12-myristate 13-acetate (PMA) in cultured Leydig tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaudhary, L.R.; Raju, V.S.; Stocco, D.M.

1987-05-01

It has been shown that addition of hCG or c-AMP to cultured Leydig tumor cells (MA-10) increases synthesis of progesterone as the major steroid. To investigate the possible involvement of protein kinase C (PK-C) in the regulation of steroid synthesis, the authors have studied the effect of PMA, an activator of PK-C, on progesterone production in MA-10 cells. The addition of PMA (100 ng/ml) stimulated steroid production whereas 4 -phorbol-12,13-didecanoate, an inactive phorbol ester, did not have any effects. Like hCG and c-AMP, PMA-stimulated progesterone production was inhibited by cycloheximide. hCG-stimulated steroid synthesis was inhibited by PMA. The addition ofmore » PMA to MA-10 Leydig cells further increased the c-AMP-stimulated progesterone production. To determine whether c-AMP has a obligatory role in the regulation of steroid production, the effect of adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (TFA), was studied on progesterone production in the presence of hCG. At lower dose (17 ng/ml) hCG-stimulated intracellular c-AMP levels and steroid production were inhibited by TFA (300 M). At higher dose of hCG (34 ng/ml) TFA did not inhibit the hCG-stimulated intracellular c-AMP levels, however, progesterone production was inhibited. Results suggest that the action of hCG, c-AMP and PMA in controlling steroidogenesis might be regulated by similar but different mechanisms.« less

Sex steroids and cervical cancer.

PubMed

Hellberg, Dan

2012-08-01

During the 19th century, studies indicated that reproductive events were involved in cervical cancer. Human papillomavirus (HPV) infection is a prerequisite for development of cancer, but co-factors, among them the action of sexual steroid hormones, are necessary. Childbirth has been an important risk factor but now probably plays a minor role in the industrialized world, where parity is low. Long-term oral contraceptive use has been thoroughly studied epidemiologically, and correlates to cervical cancer in most studies. In vitro studies on cervical cell lines transfected with HPV and animal studies indicate that sex steroid hormones are capable to induce cancer. In in vivo cervical cancer tissue studies there have been observations that endogenous progesterone in serum correlates to a negative pattern of expression of cellular and extracellular proteins, tumor markers. Immune response could be another mechanism. Estradiol might be associated with a positive pattern and high estradiol and low progesterone levels increase duration of survival in cervical cancer. Studies where treatment of compounds that influence sex steroid hormones have been given are rare and have been disappointing.

Effects of Sex Steroids on Fish Leukocytes

PubMed Central

Cabas, Isabel

2018-01-01

In vertebrates, in addition to their classically reproductive functions, steroids regulate the immune system. This action is possible mainly due to the presence of steroid receptors in the different immune cell types. Much evidence suggests that the immune system of fish is vulnerable to xenosteroids, which are ubiquitous in the aquatic environment. In vivo and in vitro assays have amply demonstrated that oestrogens interfere with both the innate and the adaptive immune system of fish by regulating the main leukocyte activities and transcriptional genes. They activate nuclear oestrogen receptors and/or G-protein coupled oestrogen receptor. Less understood is the role of androgens in the immune system, mainly due to the complexity of the transcriptional regulation of androgen receptors in fish. The aim of this manuscript is to review our present knowledge concerning the effect of sex steroid hormones and the presence of their receptors on fish leukocytes, taking into consideration that the studies performed vary as regard the fish species, doses, exposure protocols and hormones used. Moreover, we also include evidence of the probable role of progestins in the regulation of the immune system of fish. PMID:29315244

Cardiotonic Steroids Stabilize Regulator of G Protein Signaling 2 Protein Levels

PubMed Central

Sjögren, Benita; Parra, Sergio; Heath, Lauren J.; Atkins, Kevin B.; Xie, Zie-Jian

2012-01-01

Regulator of G protein signaling 2 (RGS2), a Gq-specific GTPase-activating protein, is strongly implicated in cardiovascular function. RGS2(−/−) mice are hypertensive and prone to heart failure, and several rare human mutations that accelerate RGS2 degradation have been identified among patients with hypertension. Therefore, pharmacological up-regulation of RGS2 protein levels might be beneficial. We used a β-galactosidase complementation method to screen several thousand compounds with known pharmacological functions for those that increased RGS2 protein levels. Several cardiotonic steroids (CTSs), including ouabain and digoxin, increased RGS2 but not RGS4 protein levels. CTSs increased RGS2 protein levels through a post-transcriptional mechanism, by slowing protein degradation. RGS2 mRNA levels in primary vascular smooth muscle cells were unaffected by CTS treatment, whereas protein levels were increased 2- to 3-fold. Na+/K+-ATPase was required for the increase in RGS2 protein levels, because the effect was lost in Na+/K+-ATPase-knockdown cells. Furthermore, we demonstrated that CTS-induced increases in RGS2 levels were functional and reduced receptor-stimulated, Gq-dependent, extracellular signal-regulated kinase phosphorylation. Finally, we showed that in vivo treatment with digoxin led to increased RGS2 protein levels in heart and kidney. CTS-induced increases in RGS2 protein levels and function might modify several deleterious mechanisms in hypertension and heart failure. This novel CTS mechanism might contribute to the beneficial actions of low-dose digoxin treatment in heart failure. Our results support the concept of small-molecule modulation of RGS2 protein levels as a new strategy for cardiovascular therapy. PMID:22695717

Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

PubMed Central

Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

2008-01-01

The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792

Effects of sex steroids on expression of genes regulating growth-related mechanisms in rainbow trout (Oncorhynchus mykiss).

PubMed

Cleveland, Beth M; Weber, Gregory M

2015-05-15

Effects of a single injection of 17β-estradiol (E2), testosterone (T), or 5β-dihydrotestosterone (DHT) on expression of genes central to the growth hormone (GH)/insulin-like growth factor (IGF) axis, muscle-regulatory factors, transforming growth factor-beta (TGFβ) superfamily signaling cascade, and estrogen receptors were determined in rainbow trout (Oncorhynchus mykiss) liver and white muscle tissue. In liver in addition to regulating GH sensitivity and IGF production, sex steroids also affected expression of IGF binding proteins, as E2, T, and DHT increased expression of igfbp2b and E2 also increased expression of igfbp2 and igfbp4. Regulation of this system also occurred in white muscle in which E2 increased expression of igf1, igf2, and igfbp5b1, suggesting anabolic capacity may be maintained in white muscle in the presence of E2. In contrast, DHT decreased expression of igfbp5b1. DHT and T decreased expression of myogenin, while other muscle regulatory factors were either not affected or responded similarly for all steroid treatments. Genes within the TGFβ superfamily signaling cascade responded to steroid treatment in both liver and muscle, suggesting a regulatory role for sex steroids in the ability to transmit signals initiated by TGFβ superfamily ligands, with a greater number of genes responding in liver than in muscle. Estrogen receptors were also regulated by sex steroids, with era1 expression increasing for all treatments in muscle, but only E2- and T-treatment in liver. E2 reduced expression of erb2 in liver. Collectively, these data identify how physiological mechanisms are regulated by sex steroids in a manner that promotes the disparate effects of androgens and estrogens on growth in salmonids. Published by Elsevier Inc.

Towards an understanding of the evolution of the chorioallantoic placenta: steroid biosynthesis and steroid hormone signaling in the chorioallantoic membrane of an oviparous reptile.

PubMed

Cruze, Lori; Kohno, Satomi; McCoy, Michael W; Guillette, Louis J

2012-09-01

Amniotes, mammals, reptiles, and birds form common extraembryonic membranes during development to perform essential functions, such as protection, nutrient transfer, gas exchange, and waste removal. Together with the maternal uterus, extraembryonic membranes of viviparous (live-bearing) amniotes develop as an endocrine placenta that synthesizes and responds to steroid hormones critical for development. The ability of these membranes to synthesize and respond to steroid hormone signaling has traditionally been considered an innovation of placental amniotes. However, our laboratory recently demonstrated that this ability extends to the chorioallantoic membrane (CAM) of an oviparous (egg-laying) amniote, the domestic chicken, and we hypothesized that steroidogenic extraembryonic membranes could be an evolutionarily conserved characteristic of all amniotes because of similarities in basic structure, function, and shared evolutionary ancestry. In this study, we examined steroid hormone synthesis and signaling in the CAM of another oviparous amniote, the American alligator (Alligator mississippiensis). We quantified mRNA expression of a steroidogenic factor involved in the regulation of steroidogenesis (NR5A1), the key steroidogenic enzymes involved in the synthesis of progestins (HSD3B1), androgens (CYP17A1), and estrogens (CYP19A1), and the receptors involved in the signaling of progestins (PR), androgens (AR), estrogens (ESR1 and ESR2), and glucocorticoids (GR). Furthermore, we performed protein immunolocalization for PR and ESR1. Collectively, our findings indicate that the alligator CAM has the capability to regulate, synthesize, and respond to steroid hormone signaling, thus, supporting our hypothesis that the extraembryonic membranes of Amniota share a unifying characteristic, that is, the ability to synthesize and respond to steroid hormones.

Specialized Functional Diversity and Interactions of the Na,K-ATPase

PubMed Central

Matchkov, Vladimir V.; Krivoi, Igor I.

2016-01-01

Na,K-ATPase is a protein ubiquitously expressed in the plasma membrane of all animal cells and vitally essential for their functions. A specialized functional diversity of the Na,K-ATPase isozymes is provided by molecular heterogeneity, distinct subcellular localizations, and functional interactions with molecular environment. Studies over the last decades clearly demonstrated complex and isoform-specific reciprocal functional interactions between the Na,K-ATPase and neighboring proteins and lipids. These interactions are enabled by a spatially restricted ion homeostasis, direct protein-protein/lipid interactions, and protein kinase signaling pathways. In addition to its “classical” function in ion translocation, the Na,K-ATPase is now considered as one of the most important signaling molecules in neuronal, epithelial, skeletal, cardiac and vascular tissues. Accordingly, the Na,K-ATPase forms specialized sub-cellular multimolecular microdomains which act as receptors to circulating endogenous cardiotonic steroids (CTS) triggering a number of signaling pathways. Changes in these endogenous cardiotonic steroid levels and initiated signaling responses have significant adaptive values for tissues and whole organisms under numerous physiological and pathophysiological conditions. This review discusses recent progress in the studies of functional interactions between the Na,K-ATPase and molecular microenvironment, the Na,K-ATPase-dependent signaling pathways and their significance for diversity of cell function. PMID:27252653

Growth Factors and Tension-Induced Skeletal Muscle Growth

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.

1994-01-01

The project investigated biochemical mechanisms to enhance skeletal muscle growth, and developed a computer based mechanical cell stimulator system. The biochemicals investigated in this study were insulin/(Insulin like Growth Factor) IGF-1 and Steroids. In order to analyze which growth factors are essential for stretch-induced muscle growth in vitro, we developed a defined, serum-free medium in which the differentiated, cultured avian muscle fibers could be maintained for extended periods of time. The defined medium (muscle maintenance medium, MM medium) maintains the nitrogen balance of the myofibers for 3 to 7 days, based on myofiber diameter measurements and myosin heavy chain content. Insulin and IGF-1, but not IGF-2, induced pronounced myofiber hypertrophy when added to this medium. In 5 to 7 days, muscle fiber diameters increase by 71 % to 98% compared to untreated controls. Mechanical stimulation of the avian muscle fibers in MM medium increased the sensitivity of the cells to insulin and IGF-1, based on a leftward shift of the insulin dose/response curve for protein synthesis rates. (54). We developed a ligand binding assay for IGF-1 binding proteins and found that the avian skeletal muscle cultures produced three major species of 31, 36 and 43 kD molecular weight (54) Stretch of the myofibers was found to have no significant effect on the efflux of IGF-1 binding proteins, but addition of exogenous collagen stimulated IGF-1 binding protein production 1.5 to 5 fold. Steroid hormones have a profound effect on muscle protein turnover rates in vivo, with the stress-related glucocorticoids inducing rapid skeletal muscle atrophy while androgenic steroids induce skeletal muscle growth. Exercise in humans and animals reduces the catabolic effects of glucocorticoids and may enhance the anabolic effects of androgenic steroids on skeletal muscle. In our continuing work on the involvement of exogenrus growth factors in stretch-induced avian skeletal muscle growth, we have performed experiments to determine whether mechanical stimulation of cultured avian muscle cells alters their response to anabolic steroids or glucocorticoids. In static cultures, testosterone had no effect on muscle cell growth, but 5alpha-dihydrotestosterone and the synthetic steroid stanozolol increased cell growth by up to 18% and 30%, respectively, after a three day exposure. We completed development of a new IBM-based mechanical cell stimulator system to provide greater flexibility in operating and monitoring our experiments. Our previous long term studies on myofiber growth were designed around a perfusion system of our own design. We have recently changed to performing these studies using a modified CELLCO cartridge bioreactor system Z since it has been certified as the ground-based model for the Shuttle's Space Tissue Loss (STL) F= Cell Culture Module. The current goals of this aspect of the project are three fold: 1) to design a Z cell culture system for studying avian skeletal myofiber atrophy on the Shuttle and Space Station; 0 2) to expand the use of bioreactors to cells which do not grow in either suspension or attached to the hollow fibers; and 3) to combine the bioreactor system with our computerized mechanical cell stimulator to have a better in vitro model to study tension/gravity/stretch regulation of skeletal muscle size. Preliminary studies also reported on involved : (1) how release of tension can induce rapid atrophy of tissues cultured avian skeletal muscle cells, and (2) a mechanism to transfer and maintain avian skeletal muscle organoids in modified cartridges in the Space Tissue Loss Module.

Novel calcium recognition constructions in proteins: Calcium blade and EF-hand zone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Denesyuk, Alexander I., E-mail: adenesyu@abo.fi; Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino 142290; Permyakov, Sergei E.

Metal ions can regulate various cell processes being first, second or third messengers, and some of them, especially transition metal ions, take part in catalysis in many enzymes. As an intracellular ion, Ca{sup 2+} is involved in many cellular functions from fertilization and contraction, cell differentiation and proliferation, to apoptosis and cancer. Here, we have identified and described two novel calcium recognition environments in proteins: the calcium blade zone and the EF-hand zone, common to 12 and 8 different protein families, respectively. Each of the two environments contains three distinct structural elements: (a) the well-known characteristic Dx[DN]xDG motif; (b) anmore » adjacent structurally identical segment, which binds metal ion in the same way between the calcium blade zone and the EF-hand zone; and (c) the following structurally variable segment, which distinguishes the calcium blade zone from the EF-hand zone. Both zones have sequence insertions between the last residue of the zone and calcium-binding residues in positions V or VI. The long insertion often connects the active and the calcium-binding sites in proteins. Using the structurally identical segments as an anchor, we were able to construct the classical calmodulin type EF-hand calcium-binding site out of two different calcium-binding motifs from two unrelated proteins.« less

Characterization of Steroid Receptor RNA Activator Protein Function in Modulating the Estrogen Signaling Pathway

DTIC Science & Technology

2007-05-01

Signaling Pathway 5b. GRANT NUMBER W81XWH-05-1-0245 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Yi Yan 5e. TASK NUMBER...negative control to identify proteins non-specifically precipitated. Tandem mass spectrometric analysis of immunoprecipitated samples identified a...immunoprecipitated sample and negative control. It is important to note that, SRAP was present among the remaining specifically precipitated 87 proteins. Using the

Antenatal steroid exposure in the late preterm period is associated with reduced cord blood neurotrophin-3.

PubMed

Hodyl, Nicolette A; Crawford, Tara M; McKerracher, Lorna; Lawrence, Andrew; Pitcher, Julia B; Stark, Michael J

2016-10-01

Neurotrophins are proteins critically involved in neural growth, survival and differentiation, and therefore important for fetal brain development. Reduced cord blood neurotrophins have been observed in very preterm infants (<32weeks gestation) who subsequently develop brain injury. Antenatal steroid exposure can alter neurotrophin concentrations, yet studies to date have not examined whether this occurs in the late preterm infant (33-36weeks gestation), despite increasing recognition of subtle neurodevelopmental deficits in this population. To assess the impact of antenatal steroids on cord blood neurotrophins in late preterm infants following antenatal steroid exposure. Retrospective analysis. Late preterm infants (33-36weeks; n=119) and term infants (37-41weeks; n=129) born at the Women's and Children's Hospital, Adelaide. Cord blood neurotrophin-3 (NT-3), NT-4, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) concentrations measured by ELISA. Cord blood NT-4 and NGF were increased at term compared to the late preterm period (p<0.001), while BDNF and NT-3 were not different. In the late preterm period, cord blood NT-3 was reduced when antenatal steroids were administered >24h prior to delivery (p<0.01). This study identified an association between reduced cord blood NT-3 and antenatal steroid exposure in the late preterm period. The reduced NT-3 may be a consequence of steroids inducing neuronal apoptosis, thereby reducing endogenous neuronal NT3 production, or be an action of steroids on other maternal or fetal NT-3 producing cells, which may then affect neuronal growth, differentiation and survival. Regardless of the specific mechanism, a reduction in NT-3 may have long term implications for child neurodevelopment, and emphasizes the ongoing vulnerability of the fetal brain across the full preterm period. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Perception of Plant Steroid Hormones at the Cell Surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jianming

The proposed research had two main objectives: 1) investigating the molecular mechanism by which BRs activate the BRI1-containing steroid receptor; and 2) to investigate the molecular mechanism of BRI1 function. During the course of this project, several research papers were published from other laboratories, which reported studies similar to our proposed experiments. We therefore changed our research direction and focused our research efforts on 1) molecular genetic studies of several extragenic suppressors of a weak bri1-9 mutant (which were named as EMS-mutagenized bri1 suppressor or ebs) and 2) biochemical characterization of the protein products of the cloned EBS genes. Thismore » switch turned out to be extremely successful and led to a surprising discovery that the dwarf phenotype of the well-studied bri1-9 mutant is not due to the failure of the bri1 receptor to bind the plant steroid hormone but rather caused by the retention of a structurally-imperfect but biochemically-competent bri1-9 and its subsequent degradation in the endoplasmic reticulum. This initial discovery coupled with subsequent cloning and further studies of additional EBS genes significantly increased our understanding of the protein quality control mechanisms in plants, a severely under-studied research topic in plant biology.« less

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

PubMed

Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

2002-08-16

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Dynamics of subcellular compartmentalization of steroid receptors in living cells as a strategic screening method to determine the biological impact of suspected endocrine disruptors.

PubMed

Tyagi, Rakesh Kumar

2003-04-01

Although a number of screening methods being used for identifying potential endocrine disruptors have generated a wealth of information, a search for alternative combination of methods is still needed to overcome experimental artefacts. There are no generally accepted or validated screening methods for monitoring and studying impact of environmental endocrine disruptors. Also, no single assay can accurately predict all the deleterious effects of endocrine disruptors. For this reason various environmental protection agencies, mainly European and US, have urged that a battery of tests in current use need to be designed to assess their adequacy in detecting the effects of endocrine disruptors. Some details about endocrine disruptors and screening programs can be found at http://www.epa.gov/scipoly/oscpendo/whatis.htm. Several studies in recent years have used fusion proteins between steroid receptors (estrogen, androgen, progesterone, etc.) and green fluorescent protein (GFP) that can serve as an alternative potent screening method to study intracellular dynamics of receptors in living cells. An approach employing nucleocytoplasmic trafficking of steroid receptors as a parameter in response to potential xenobiotic chemicals in living cells may prove to be promising in terms of being direct, fast, reliable, simple and inexpensive. Copyright 2003 Elsevier Science Ltd.

Skeletal muscle metabolism in hypokinetic rats

NASA Technical Reports Server (NTRS)

Tischler, M. E.

1984-01-01

Muscle growth, protein metabolism, and amino acid metabolism were studied in various groups of rats. Certain groups were adrenaliectomized; some rats were suspended while others (the controls) were weight bearing. Results show that: (1) metabolic changes in the extensor digitorum longus muscle of suspended rats are due primarily to increased circulating glucocorticoids; (2) metabolic changes in the soleus muscle due to higher steroid levels are probably potentiated by greater numbers of steroid receptors; and (3) not all metabolic responses of the soleus muscle to unloading are due to the elevated levels of glucocorticoids or the increased sensitivity of this muscle to these hormones.

Steroid Receptor Coactivator-interacting Protein (SIP) Inhibits Caspase-independent Apoptosis by Preventing Apoptosis-inducing Factor (AIF) from Being Released from Mitochondria*

PubMed Central

Wang, Dandan; Liang, Jing; Zhang, Yu; Gui, Bin; Wang, Feng; Yi, Xia; Sun, Luyang; Yao, Zhi; Shang, Yongfeng

2012-01-01

Apoptosis-inducing factor (AIF) is a caspase-independent death effector. Normally residing in the mitochondrial intermembrane space, AIF is released and translocated to the nucleus in response to proapoptotic stimuli. Nuclear AIF binds to DNA and induces chromatin condensation and DNA fragmentation, characteristics of apoptosis. Until now, it remained to be clarified how the mitochondrial-nuclear translocation of AIF is regulated. Here we report that steroid receptor coactivator-interacting protein (SIP) interacts directly with AIF in mitochondria and specifically inhibits caspase-independent and AIF-dependent apoptosis. Challenging cells with apoptotic stimuli leads to rapid degradation of SIP, and subsequently AIF is liberated from mitochondria and translocated to the nucleus to induce apoptosis. Together, our data demonstrate that SIP is a novel regulator in caspase-independent and AIF-mediated apoptosis. PMID:22371500

Effects of Protein, Lipids, and Surfactants on the Antimicrobial Activity of Synthetic Steroids

PubMed Central

Smith, Rodney F.; Shay, Donald E.; Doorenbos, Norman J.

1963-01-01

Three 4-azacholestanes and two A-norcholestanes were inactivated by 10 and 20% bovine serum and by 1.0, 2.5, and 5.0% sheep blood. The five compounds exhibited hemolytic properties when tested with 2% sheep blood and 2% human blood. These cholestanes inhibited Streptococcus pyogenes and were completely inactivated by 0.1% lecithin. Tween 80 was comparable to lecithin in causing the inactivation of steroids; 1% polyethylene glycol-4000 was inert; 1% Tween 20 and 1.0% Span 20 caused the inactivation of 3β,4-dimethyl-4-aza-5α-cholestane (ND-307). The sodium salts of four fatty acids, oleate, stearate, deoxycholate, and lauryl sulfate (0.1 to 1.0 mg/ml), effectively interfered with the action of ND-307. The steroids appear to have some properties similar to those of antimicrobial surfactants of the cationic type but have certain distinct features. PMID:14075055

Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors.

PubMed

Natale, Christopher A; Duperret, Elizabeth K; Zhang, Junqian; Sadeghi, Rochelle; Dahal, Ankit; O'Brien, Kevin Tyler; Cookson, Rosa; Winkler, Jeffrey D; Ridky, Todd W

2016-04-26

The association between pregnancy and altered cutaneous pigmentation has been documented for over two millennia, suggesting that sex hormones play a role in regulating epidermal melanocyte (MC) homeostasis. Here we show that physiologic estrogen (17β-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that normal primary human MCs lack classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G protein-coupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics.

Modulation of the cytosolic androgen receptor in striated muscle by sex steroids

NASA Technical Reports Server (NTRS)

Rance, N. E.; Max, S. E.

1982-01-01

The influence of orchiectomy (GDX) and steroid administration on the level of the cytosolic androgen receptor in the rat levator ani muscle and in rat skeletal muscles (tibialis anterior and extensor digitorum longus) was studied. Androgen receptor binding to muscle cytosol was measured using H-3 methyltrienolone (R1881) as ligand, 100 fold molar excess unlabeled R1881 to assess nonspecific binding, and 500 fold molar excess of triamcinolone acetonide to prevent binding to glucocorticoid and progestin receptors. Results demonstrate that modification of the levels of sex steroids can alter the content of androgen receptors of rat striated muscle. Data suggest that: (1) cytosolic androgen receptor levels increase after orchiectomy in both levator ani muscle and skeletal muscle; (2) the acute increase in receptor levels is blocked by an inhibitor of protein synthesis; and (3) administration of estradiol-17 beta to castrated animals increases receptor binding in levator ani muscle but not in skeletal muscle.

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

PubMed Central

Riggs, Daniel L.; Roberts, Patricia J.; Chirillo, Samantha C.; Cheung-Flynn, Joyce; Prapapanich, Viravan; Ratajczak, Thomas; Gaber, Richard; Picard, Didier; Smith, David F.

2003-01-01

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52. PMID:12606580

Separation of an associated 90K heat shock protein from the glucocorticoid receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller-Diener, A.; Kirsch, T.; Grove, B.

1986-05-01

A 90K heat shock protein(HSP), observed to copurify with the glucocorticoid receptor(GR), can be separated from the complex by 2 methods, allowing investigation of the role of HSP on kinase activity that was previously reported to be inherent to purified activated GR. Na/sub 2/MoO/sub 4/ stabilized unactivated rat hepatic GR complexes have been purified to >10,000-fold using a purification scheme that involves batchwise treatment of cytosol with phosphocellulose/DNAcellulose, elution from an affinity resin, gel filtration and ion exchange chromatography. Samples were subjected to 10-20% gradient SDS-PAGE. Proteins were transferred to nitrocellulose and blotted against monoclonal antibodies to GR(3A6), HSP ormore » nonspecific IgM/G. Immunoblots indicated that HSP was separated from unactivated GR complexes at the affinity step prior to elution of GR with active steroid. GR eluted from the resin with /sup 3/H Triamcinolone acetonide or /sup 3/H Dexamethasone mesylate had an apparent M/sub r/ = 94-96,000 for the steroid binding subunit and is recognized by 3A6. Purification of GR minus the affinity step resulted in copurification of HSP throughout the procedure. However, after Sephadex G75 filtration and subsequent incubation at 25/sup 0/C, 30 min., HSP was separated from activated (DNA binding) GR on DEAE cellulose-52. HSP did not enhance or inhibit /sup 32/P incorporation of the 94K steroid binding subunit nor did it affect phosphorylation of histones by GR.« less

Fusion competent synaptic vesicles persist upon active zone disruption and loss of vesicle docking

PubMed Central

Wang, Shan Shan H.; Held, Richard G.; Wong, Man Yan; Liu, Changliang; Karakhanyan, Aziz; Kaeser, Pascal S.

2016-01-01

In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane. PMID:27537483

Synergistic Activation of Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis by Retinoids: Involvement of cAMP/PKA Signaling

PubMed Central

Manna, Pulak R.; Slominski, Andrzej T.; King, Steven R.; Stetson, Cloyce L.

2014-01-01

Both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the action of retinoids that play important roles in reproductive development and function, as well as steroidogenesis. Regulation of steroid biosynthesis is principally mediated by the steroidogenic acute regulatory protein (StAR); however, the modes of action of retinoids in the regulation of steroidogenesis remain obscure. In this study we demonstrate that all-trans retinoic acid (atRA) enhances StAR expression, but not its phosphorylation (P-StAR), and progesterone production in MA-10 mouse Leydig cells. Activation of the protein kinase A (PKA) cascade, by dibutyrl-cAMP or type I/II PKA analogs, markedly increased retinoid-responsive StAR, P-StAR, and steroid levels. Targeted silencing of endogenous RARα and RXRα, with small interfering RNAs, resulted in decreases in 9-cis RA-stimulated StAR and progesterone levels. Truncation of and mutational alterations in the 5′-flanking region of the StAR gene demonstrated the importance of the −254/−1-bp region in retinoid responsiveness. An oligonucleotide probe encompassing an RXR/liver X receptor recognition motif, located within the −254/−1-bp region, specifically bound MA-10 nuclear proteins and in vitro transcribed/translated RXRα and RARα in EMSAs. Transcription of the StAR gene in response to atRA and dibutyrl-cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of cAMP response-element binding protein (CREB). Chromatin immunoprecipitation studies revealed the association of phosphorylation of CREB, CREB binding protein, RXRα, and RARα to the StAR promoter. Further studies elucidated that hormone-sensitive lipase plays an important role in atRA-mediated regulation of the steroidogenic response that involves liver X receptor signaling. These findings delineate the molecular events by which retinoids influence cAMP/PKA signaling and provide additional and novel insight into the regulation of StAR expression and steroidogenesis in mouse Leydig cells. PMID:24265455

Gonadal steroids regulate the expression of aggrecanases in human endometrial stromal cells in vitro

PubMed Central

Wen, Jiadi; Zhu, Hua; Leung, Peter CK

2013-01-01

The human endometrium undergoes cyclic change during each menstrual cycle in response to gonadal steroids. Proteolysis of endometrial extracellular matrix (ECM) is necessary to prepare this dynamic tissue for pregnancy. Proteolytic enzymes such as matrix metalloproteinase (MMP) and closely related a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been assigned key roles in the highly regulated cyclic remodelling of the endometrial ECM. We have previously shown that ADAMTS-1 undergoes spatiotemporal changes in human endometrial stromal cells under the regulation of gonadal steroids. This suggests that other ADAMTS subtypes, known as aggrecanases, may contribute to the ECM remodelling events that occur in female physiological cycles and in preparation for pregnancy. To determine whether progesterone (P4), 17β-estradiol (E2), or dihydrotestosterone (DHT), alone or in combination, are capable of regulating ADAMTS-4, -5, -8 or -9 expression in human endometrial stromal cells in vitro. Real-time quantitative PCR and Western blot analysis were used to measure ADAMTSs mRNA and protein levels in primary cultures of human endometrial stromal cells (n = 12). P4, DHT but not E2 have regulatory effects on ADAMTS-8, -9 and -5 expression. Combined treatment with gonadal steroids did not show any synergistic or antagonistic effects. However, the synthetic steroid antagonists RU486 and hydroxyflutamide specifically inhibited the P4- or DHT-mediated regulatory effects on ADAMTS expression. These studies provide evidence that the regulation of aggrecanases by gonadal steroids in human endometrial stromal cells may play an important role during decidualization. PMID:23947778

Reproductive endocrinology in chondrichthyans: the present and the future.

PubMed

Awruch, C A

2013-10-01

The class Chondrichthyes, that includes Elasmobranchii and Holocephali, is a diverse group of fish occupying a key position at the base of vertebrate evolution. Their evolutionary success is greatly attributed to their wide range of reproductive strategies controlled by different endocrine mechanics. As in other vertebrates, hormonal control of reproduction in chondrichthyans is mediated by the neuropeptide gonadotropin-releasing hormone (GnRH) that regulates the brain control of gonadal activity via a hypothalamus-pituitary-gonadal (HPG) axis. Chondrichthyans lack of a direct vascular supply from the hypothalamus to the zone of the pituitary where the gonadotropic activity resides, thus transport between these two zones likely occurs via the general circulation. In the brain of elasmobranchs, two groups of GnRH, GnRH-I and GnRH-II were identified, and the presence of two immunoreactive gonadotropins similar to the luteinising (LH) and follicle stimulating (FSH) hormones was identified in the pituitary. In holocephalans, only GnRH-II has been confirmed, and while gonadotropin activity has been found in the buccal pituitary lobe, the presence of gonadotropin receptors in the gonads remains unknowns. The diversity of reproductive strategies display by chondrichthyans makes it difficult to generalize the control of gametogenesis and steroidogenesis; however, some general patterns emerge. In both sexes, androgens and estrogens are the main steroids during gonadal growth; while progestins have maturational activity. Androgens also form the precursors for estrogen steroid production. Estrogens stimulate the hepatic synthesis of yolk and stimulate the development of different part of the reproductive tract in females. The role of other gonadal steroids may play in chondrichthyan reproduction remains largely unknown. Future work should concentrate in filling the gaps into the current knowledge of the HPG axis regulation, and the use of reproductive endocrinology as a non-lethal technique for management of chondrichthyan populations. Copyright © 2013 Elsevier Inc. All rights reserved.

Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Department of Oncology, Georgetown University School of Medicine, Washington, DC 20057

2005-08-15

The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increasedmore » ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids.« less

Circulating gonadotropins and ovarian adiponectin system are modulated by acupuncture independently of sex steroid or β-adrenergic action in a female hyperandrogenic rat model of polycystic ovary syndrome.

PubMed

Maliqueo, Manuel; Benrick, Anna; Alvi, Asif; Johansson, Julia; Sun, Miao; Labrie, Fernand; Ohlsson, Claes; Stener-Victorin, Elisabet

2015-09-05

Acupuncture with combined manual and low-frequency electrical stimulation, or electroacupuncture (EA), reduces endocrine and reproductive dysfunction in women with polycystic ovary syndrome (PCOS), likely by modulating sympathetic nerve activity or sex steroid synthesis. To test this hypothesis, we induced PCOS in rats by prepubertal implantation of continuous-release letrozole pellets (200 µg/day) or vehicle. Six weeks later, rats were treated for 5-6 weeks with low-frequency EA 5 days/week, subcutaneous injection of 17β-estradiol (2.0 µg) every fourth day, or a β-adrenergic blocker (propranolol hydrochloride, 0.1 mg/kg) 5 days/week. Letrozole controls were handled without needle insertion or injected with sesame oil every fourth day. Estrous cyclicity, ovarian morphology, sex steroids, gonadotropins, insulin-like growth factor I, bone mineral density, and gene and protein expression in ovarian tissue were measured. Low-frequency EA induced estrous-cycle changes, decreased high levels of circulating luteinizing hormone (LH) and the LH/follicle-stimulating hormone (FSH) ratio, decreased high ovarian gene expression of adiponectin receptor 2, and increased expression of adiponectin receptor 2 protein and phosphorylation of ERK1/2. EA also increased cortical bone mineral density. Propranolol decreased ovarian expression of Foxo3, Srd5a1, and Hif1a. Estradiol decreased circulating LH, induced estrous cycle changes, and decreased ovarian expression of Adipor1, Foxo3, and Pik3r1. Further, total bone mineral density was higher in the letrozole-estradiol group. Thus, EA modulates the circulating gonadotropin levels independently of sex steroids or β-adrenergic action and affects the expression of ovarian adiponectin system. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Altered expression of epithelial mesenchymal transition and pluripotent associated markers by sex steroid hormones in human embryonic stem cells.

PubMed

Jeon, So-Ye; Hwang, Kyung-A; Kim, Cho-Won; Jeung, Eui-Bae; Choi, Kyung-Chul

2017-07-01

Embryonic stem (ES) cells are pluripotent stem cells derived from a developmental stage of pre‑implanted embryos. The present study investigated the effect of female sex steroid hormones on the characteristics of human ES cells by using a feeder‑free culture protocol. In a feeder‑free condition without sex hormones, human ES cells assumed the form of tightly packed cells that grow in a monolayer. The cells had clean and defined edges with no evidence of differentiation and expressed several markers specific for undifferentiated ES cells including POU class 5 homeobox 1 (POU5F1), sex determining region Y‑box 2 (SOX2) and NANOG homeobox (NANOG). It was then investigated if female sex steroid hormones including 17β‑estradiol (E2) and progesterone (P4) altered the protein expression of epithelial-mesenchymal transition (EMT) associated markers in addition to pluripotency markers including POU5F1, SOX2 and NANOG in human ES cells. The protein expression levels of N‑cadherin, Snail and Slug were increased while E‑cadherin expression was decreased by treatment of E2 or P4, and the expression levels of POU5F1, SOX2 and NANOG were decreased by the treatment of E2 or P4. When E2 and P4 were treated in combination with an estrogen receptor inhibitor (ICI 182,780) and progesterone receptor inhibitor (RU486) respectively, their effects on EMT and pluripotency of ES cells were restored to control levels. The results suggested that E2 and P4 may regulate EMT and pluripotency of human ES cells by mediating their receptors. The present study may aid in the understanding of the role of sex steroid hormones in the cellular biology of human ES cells.

Supraphysiological doses of performance enhancing anabolic-androgenic steroids exert direct toxic effects on neuron-like cells

PubMed Central

Basile, John R.; Binmadi, Nada O.; Zhou, Hua; Yang, Ying-Hua; Paoli, Antonio; Proia, Patrizia

2013-01-01

Anabolic-androgenic steroids (AAS) are lipophilic hormones often taken in excessive quantities by athletes and bodybuilders to enhance performance and increase muscle mass. AAS exert well known toxic effects on specific cell and tissue types and organ systems. The attention that androgen abuse has received lately should be used as an opportunity to educate both athletes and the general population regarding their adverse effects. Among numerous commercially available steroid hormones, very few have been specifically tested for direct neurotoxicity. We evaluated the effects of supraphysiological doses of methandienone and 17-α-methyltestosterone on sympathetic-like neuron cells. Vitality and apoptotic effects were analyzed, and immunofluorescence staining and western blot performed. In this study, we demonstrate that exposure of supraphysiological doses of methandienone and 17-α-methyltestosterone are toxic to the neuron-like differentiated pheochromocytoma cell line PC12, as confirmed by toxicity on neurite networks responding to nerve growth factor and the modulation of the survival and apoptosis-related proteins ERK, caspase-3, poly (ADP-ribose) polymerase and heat-shock protein 90. We observe, in contrast to some previous reports but in accordance with others, expression of the androgen receptor (AR) in neuron-like cells, which when inhibited mitigated the toxic effects of AAS tested, suggesting that the AR could be binding these steroid hormones to induce genomic effects. We also note elevated transcription of neuritin in treated cells, a neurotropic factor likely expressed in an attempt to resist neurotoxicity. Taken together, these results demonstrate that supraphysiological exposure to the AAS methandienone and 17-α-methyltestosterone exert neurotoxic effects by an increase in the activity of the intrinsic apoptotic pathway and alterations in neurite networks. PMID:23675320

GH improves spatial memory and reverses certain anabolic androgenic steroid-induced effects in intact rats.

PubMed

Grönbladh, Alfhild; Johansson, Jenny; Nöstl, Anatole; Nyberg, Fred; Hallberg, Mathias

2013-01-01

GH has previously been shown to promote cognitive functions in GH-deficient rodents. In this study we report the effects of GH on learning and memory in intact rats pretreated with the anabolic androgenic steroid nandrolone. Male Wistar rats received nandrolone decanoate (15 mg/kg) or peanut oil every third day for 3 weeks and were subsequently treated with recombinant human GH (1.0 IU/kg) or saline for 10 consecutive days. During the GH/saline treatment spatial learning and memory were tested in the Morris water maze (MWM). Also, plasma levels of IGF1 were assessed and the gene expression of the GH receptors (Ghr), Igf1 and Igf2, in hippocampus and frontal cortex was analyzed. The results demonstrated a significant positive effect of GH on memory functions and increased gene expression of Igf1 in the hippocampus was found in the animals treated with GH. In addition, GH was demonstrated to increase the body weight gain and was able to attenuate the reduced body weight seen in nandrolone-treated animals. In general, the rats treated with nandrolone alone did not exhibit any pronounced alteration in memory compared with controls in the MWM, and in many cases GH did not induce any alteration. Regarding target zone crossings, considered to be associated with spatial memory, the difference between GH- and steroid-treated animals was significant and administration of GH improved this parameter in the latter group. In conclusion, GH improves spatial memory in intact rats and can reverse certain effects induced by anabolic androgenic steroid.

Inhibition of transcription affects synthesis of steroidogenic acute regulatory protein and steroidogenesis in MA-10 mouse Leydig tumor cells.

PubMed

Clark, B J; Combs, R; Hales, K H; Hales, D B; Stocco, D M

1997-11-01

Hormonal induction of steroidogenesis in the adrenal and gonads is dependent on the synthesis and function of the steroidogenic acute regulatory protein (StAR). As a first approach to investigate the role of translation in the control of StAR expression, we examined StAR protein synthesis and steroid production in MA-10 mouse Leydig tumor cells in the presence of the transcriptional inhibitor, actinomycin D. We show that human CG (hCG)-induced StAR synthesis, as determined by radiolabeling MA-10 cells with [35S]methionine and immunoprecipitation of StAR, is blocked by actinomycin D. The rate of hCG-stimulated progesterone production is also decreased, but not completely blocked, suggesting a possible StAR-independent mechanism that may contribute approximately 10-20% of the acute steroidogenic potential of the cells. When MA-10 cells were pretreated with hCG to increase StAR messenger RNA levels and then the proteins radiolabeled in the presence of hCG or hCG plus actinomycin D, no difference was observed in the amount of the 30-kDa StAR protein synthesized. However, a 50% increase in the precursor form of StAR protein was detected with hCG treatment alone. These data suggest that ongoing StAR protein synthesis is not inhibited by actinomycin D, but that continued synthesis requires transcriptional activity. Progesterone production was inhibited by actinomycin D in the hCG-pretreated cells, supporting the proposal that maintaining StAR protein synthesis is required for optimal steroid production in MA-10 mouse Leydig tumor cells.

ELKS active zone proteins as multitasking scaffolds for secretion

PubMed Central

Held, Richard G.

2018-01-01

Synaptic vesicle exocytosis relies on the tethering of release ready vesicles close to voltage-gated Ca2+ channels and specific lipids at the future site of fusion. This enables rapid and efficient neurotransmitter secretion during presynaptic depolarization by an action potential. Extensive research has revealed that this tethering is mediated by an active zone, a protein dense structure that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Although roles of individual active zone proteins in exocytosis are in part understood, the molecular mechanisms that hold the protein scaffold at the active zone together and link it to the presynaptic plasma membrane have remained unknown. This is largely due to redundancy within and across scaffolding protein families at the active zone. Recent studies, however, have uncovered that ELKS proteins, also called ERC, Rab6IP2 or CAST, act as active zone scaffolds redundant with RIMs. This redundancy has led to diverse synaptic phenotypes in studies of ELKS knockout mice, perhaps because different synapses rely to a variable extent on scaffolding redundancy. In this review, we first evaluate the need for presynaptic scaffolding, and we then discuss how the diverse synaptic and non-synaptic functional roles of ELKS support the hypothesis that ELKS provides molecular scaffolding for organizing vesicle traffic at the presynaptic active zone and in other cellular compartments. PMID:29491150

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

PubMed

Witzel, Katja; Matros, Andrea; Møller, Anders L B; Ramireddy, Eswarayya; Finnie, Christine; Peukert, Manuela; Rutten, Twan; Herzog, Andreas; Kunze, Gotthard; Melzer, Michael; Kaspar-Schoenefeld, Stephanie; Schmülling, Thomas; Svensson, Birte; Mock, Hans-Peter

2018-06-01

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity. © 2018 John Wiley & Sons Ltd.

VGKC-complex/LGI1-antibody encephalitis: clinical manifestations and response to immunotherapy.

PubMed

Shin, Yong-Won; Lee, Soon-Tae; Shin, Jung-Won; Moon, Jangsup; Lim, Jung-Ah; Byun, Jung-Ick; Kim, Tae-Joon; Lee, Keon-Joo; Kim, Young-Su; Park, Kyung-Il; Jung, Keun-Hwa; Lee, Sang Kun; Chu, Kon

2013-12-15

Leucine-rich glioma inactivated 1 (LGI1) was recently identified as a target protein in autoimmune synaptic encephalitis, a rare condition associated with autoantibodies against structures in the neuronal synapse. Studies dealing with LGI1 are small in number and the various outcomes of different therapeutic regimens are not well studied. Here, we analyzed clinical characteristics of 14 patients with LGI1 antibodies, and outcomes according to therapeutic strategies. Most patients exhibited abnormal brain positron emission tomography and that patients treated with steroids alone were more likely to relapse and had less favorable outcomes than those treated with steroids and intravenous immunoglobulins. © 2013.

Estradiol regulates the insulin-like growth factor-I (IGF-I) signalling pathway: A crucial role of phosphatidylinositol 3-kinase (PI 3-kinase) in estrogens requirement for growth of MCF-7 human breast carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, Laurence; Legay, Christine; Adriaenssens, Eric

2006-12-01

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. With a view to investigating the molecular mechanisms implicated, we studied the effect of estradiol on the expression of proteins implicated in the insulin-like growth factor signalling pathway. Estradiol dose- and time-dependently increased the expression of insulin receptor substrate-1 and the p85/p110 subunits of phosphatidylinositol 3-kinase but did not change those of ERK2 and Akt/PKB. ICI 182,780 did not inhibit estradiol-induced IRS-1 and p85 expression. Moreover, two distinct estradiol-BSA conjugate compounds were as effective as estradiol in inducing IRS-1 and p85/p110more » expression indicating the possible implication of an estradiol membrane receptor. Comparative analysis of steroids-depleted and steroids-treated cells showed that IGF-I only stimulates cell growth in the latter condition. Nevertheless, expression of a constitutively active form of PI 3-kinase in steroid-depleted cells triggers proliferation. These results demonstrate that estradiol positively regulates essential proteins of the IGF signalling pathway and put in evidence that phosphatidylinositol 3-kinase plays a central role in the synergistic pro-proliferative action of estradiol and IGF-I.« less

Effluents from oil production activities contain chemicals that interfere with normal function of intra- and extra-cellular estrogen binding proteins.

PubMed

Tollefsen, Knut-Erik; Finne, Eivind Farmen; Romstad, Randi; Sandberg, Cecilie

2006-07-01

Some environmental pollutants have the ability to alter the endocrine function in fish through interaction with the estrogen receptor (ER). Many of these chemicals are also able to interfere with the endocrine system through other mechanisms of action, however. The plasma sex steroid-binding protein (SBP), which is involved in regulating circulating levels of endogenous sex steroids, has recently been proposed to contribute to pollutant induced disruption of endocrine homeostasis. The objective of the present work was to determine whether industrial effluents contain chemicals that are able to modulate the endocrine system through interference with the function of the ER and SBP using in vitro biological assays (bioassays) from rainbow trout. The results show that solid phase extracts of process water (produced water) from an oil production facility in the North Sea and a land-based oil refinery contain chemicals that are able to induce estrogenic effects as well as displace natural sex steroid 17beta-estradiol from the SBP. The bioactive chemicals were found to be partly resistant to biological degradation, but the identity of the chemicals was not determined. The alkylphenol 4-tert-butylphenol, which is known to occur in effluents from various oil production facilities, was found to be estrogenic and displace 17beta-estradiol from the SBP and may thus contribute to the observed endocrine disrupting activity.

Proteomic analysis indicates massive changes in metabolism prior to the inhibition of growth and photosynthesis of grapevine (Vitis vinifera L.) in response to water deficit

PubMed Central

2013-01-01

Background Cabernet Sauvignon grapevines were exposed to a progressive, increasing water defict over 16 days. Shoot elongation and photosynthesis were measured for physiological responses to water deficit. The effect of water deficit over time on the abundance of individual proteins in growing shoot tips (including four immature leaves) was analyzed using nanoflow liquid chromatography - tandem mass spectrometry (nanoLC-MS/MS). Results Water deficit progressively decreased shoot elongation, stomatal conductance and photosynthesis after Day 4; 2277 proteins were identified by shotgun proteomics with an average CV of 9% for the protein abundance of all proteins. There were 472 out of 942 (50%) proteins found in all samples that were significantly affected by water deficit. The 472 proteins were clustered into four groups: increased and decreased abundance of early- and late-responding protein profiles. Vines sensed the water deficit early, appearing to acclimate to stress, because the abundance of many proteins changed before decreases in shoot elongation, stomatal conductance and photosynthesis. Predominant functional categories of the early-responding proteins included photosynthesis, glycolysis, translation, antioxidant defense and growth-related categories (steroid metabolism and water transport), whereas additional proteins for late-responding proteins were largely involved with transport, photorespiration, antioxidants, amino acid and carbohydrate metabolism. Conclusions Proteomic responses to water deficit were dynamic with early, significant changes in abundance of proteins involved in translation, energy, antioxidant defense and steroid metabolism. The abundance of these proteins changed prior to any detectable decreases in shoot elongation, stomatal conductance or photosynthesis. Many of these early-responding proteins are known to be regulated by post-transcriptional modifications such as phosphorylation. The proteomics analysis indicates massive and substantial changes in plant metabolism that appear to funnel carbon and energy into antioxidant defenses in the very early stages of plant response to water deficit before any significant injury. PMID:23514573

Reproductive adverse outcome pathways for chemical inhibitors of steroid synthesis in fish

EPA Science Inventory

A key physiological process controlling reproductive success of oviparous vertebrates (fish, amphibian, reptiles, birds) involves production of the egg yolk protein precursor vitellogenin (VTG). VTG production is an estrogen receptor (ER)-mediated process that, in females, is con...

ESTROGEN INDUCED VITELLOGENIN MRNA AND PROTEIN IN SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)

EPA Science Inventory

Many environmentally persistent xenobiotic chemicals appear to disrupt normal endocrine function by acting as ligands for endogenous steroid receptors, including the estrogen receptor. Xenobiotics that bind to the estrogen receptor may elicit several effects, one of which is acti...

Vestigialization of an Allosteric Switch: Genetic and Structural Mechanisms for the Evolution of Constitutive Activity in a Steroid Hormone Receptor

PubMed Central

Bridgham, Jamie T.; Keay, June; Ortlund, Eric A.; Thornton, Joseph W.

2014-01-01

An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs), a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors) activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER), and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become “stuck” in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large-effect mutations to trigger a profound evolutionary change in the protein's function and shaped the genetics of evolutionary reversibility. PMID:24415950

Hair cortisol and progesterone detection in dairy cattle: interrelation with physiological status and milk production.

PubMed

Tallo-Parra, O; Carbajal, A; Monclús, L; Manteca, X; Lopez-Bejar, M

2018-07-01

Hair cortisol concentrations (HCCs) and hair progesterone concentrations (HPCs) allow monitoring long-term retrospective steroid levels. However, there are still gaps in the knowledge of the mechanisms of steroid deposition in hair and its potential application in dairy cattle research. This study aimed to evaluate the potential uses of hair steroid determinations by studying the interrelations between HCC, HPC, physiological data from cows, and their milk production and quality. Cortisol and progesterone concentrations were analyzed in hair from 101 milking Holstein Friesian cows in a commercial farm. Physiological data were obtained from the 60 d prior to hair collection. Moreover, productive data from the month when hair was collected and the previous one were also obtained as well as at 124 d after hair sampling. Significant but weak correlations were found between HCC and HPC (r = 0.25, P < 0.0001) and between HPC and age (r = 0.06, P = 0.0133). High HCC were associated with low milk yields from the 2 previous months to hair sampling (P = 0.0396) and during the whole lactation (P < 0.0001). High HCC were also related to high somatic cell count (P = 0.0241). No effect of HCC on fat or protein content was detected. No significant correlations were detected between hair steroid concentrations and pregnancy status, days of gestation, parturition category (primiparous vs multiparous), number of lactations or days in milk. The relationship between physiological variables and HCC or HPC could depend on the duration of the time period over which hair accumulates hormones. Steroid concentrations in hair present high variability between individuals but are a potential tool for dairy cattle welfare and production research by providing a useful and practical tool for long-term steroid monitoring. Copyright © 2018 Elsevier Inc. All rights reserved.

Long-Term Recordings of Arcuate Nucleus Kisspeptin Neurons Reveal Patterned Activity That Is Modulated by Gonadal Steroids in Male Mice.

PubMed

Vanacker, Charlotte; Moya, Manuel Ricu; DeFazio, R Anthony; Johnson, Michael L; Moenter, Suzanne M

2017-10-01

Pulsatile release of gonadotropin-releasing hormone (GnRH) is key to fertility. Pulse frequency is modulated by gonadal steroids and likely arises subsequent to coordination of GnRH neuron firing activity. The source of rhythm generation and the site of steroid feedback remain critical unanswered questions. Arcuate neurons that synthesize kisspeptin, neurokinin B, and dynorphin (KNDy) may be involved in both of these processes. We tested the hypotheses that action potential firing in KNDy neurons is episodic and that gonadal steroids regulate this pattern. Targeted extracellular recordings were made of green fluorescent protein-identified KNDy neurons in brain slices from adult male mice that were intact, castrated, or castrated and treated with estradiol or dihydrotestosterone (DHT). KNDy neurons exhibited marked peaks and nadirs in action potential firing activity during recordings lasting 1 to 3.5 hours. Peaks, identified by Cluster analysis, occurred more frequently in castrated than intact mice, and either estradiol or DHT in vivo or blocking neurokinin type 3 receptor in vitro restored peak frequency to intact levels. The frequency of peaks in firing rate and estradiol regulation of this frequency is similar to that observed for GnRH neurons, whereas DHT suppressed firing in KNDy but not GnRH neurons. We further examined the patterning of action potentials to identify bursts that may be associated with increased neuromodulator release. Burst frequency and duration are increased in castrated compared with intact and steroid-treated mice. The observation that KNDy neurons fire in an episodic manner that is regulated by steroid feedback is consistent with a role for these neurons in GnRH pulse generation and regulation. Copyright © 2017 Endocrine Society.

Gonadal steroids regulate the expression of aggrecanases in human endometrial stromal cells in vitro.

PubMed

Wen, Jiadi; Zhu, Hua; Leung, Peter C K

2013-10-01

The human endometrium undergoes cyclic change during each menstrual cycle in response to gonadal steroids. Proteolysis of endometrial extracellular matrix (ECM) is necessary to prepare this dynamic tissue for pregnancy. Proteolytic enzymes such as matrix metalloproteinase (MMP) and closely related a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been assigned key roles in the highly regulated cyclic remodelling of the endometrial ECM. We have previously shown that ADAMTS-1 undergoes spatiotemporal changes in human endometrial stromal cells under the regulation of gonadal steroids. This suggests that other ADAMTS subtypes, known as aggrecanases, may contribute to the ECM remodelling events that occur in female physiological cycles and in preparation for pregnancy. To determine whether progesterone (P4), 17β-estradiol (E2), or dihydrotestosterone (DHT), alone or in combination, are capable of regulating ADAMTS-4, -5, -8 or -9 expression in human endometrial stromal cells in vitro. Real-time quantitative PCR and Western blot analysis were used to measure ADAMTSs mRNA and protein levels in primary cultures of human endometrial stromal cells (n = 12). P4, DHT but not E2 have regulatory effects on ADAMTS-8, -9 and -5 expression. Combined treatment with gonadal steroids did not show any synergistic or antagonistic effects. However, the synthetic steroid antagonists RU486 and hydroxyflutamide specifically inhibited the P4- or DHT-mediated regulatory effects on ADAMTS expression. These studies provide evidence that the regulation of aggrecanases by gonadal steroids in human endometrial stromal cells may play an important role during decidualization. © 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

C/EBPβ (CCAAT/enhancer-binding protein β) mediates progesterone production through transcriptional regulation in co-operation with SF-1 (steroidogenic factor-1).

PubMed

Mizutani, Tetsuya; Ju, Yunfeng; Imamichi, Yoshitaka; Osaki, Tsukasa; Yazawa, Takashi; Kawabe, Shinya; Ishikane, Shin; Matsumura, Takehiro; Kanno, Masafumi; Kamiki, Yasue; Kimura, Kohei; Minamino, Naoto; Miyamoto, Kaoru

2014-06-15

The transcription factor SF-1 (steroidogenic factor-1) is a master regulator of steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPβ working together with SF-1 are poorly understood. C/EBPβ knockdown attenuated cAMP-induced progesterone production in granulosa tumour-derived KGN cells by altering STAR (steroidogenic acute regulatory protein), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPβ-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPβ-responsive regions were found in each promoter and C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

Presynaptic Active Zone Density during Development and Synaptic Plasticity.

PubMed

Clarke, Gwenaëlle L; Chen, Jie; Nishimune, Hiroshi

2012-01-01

Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density) during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs), the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS), active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated.

Presynaptic Active Zone Density during Development and Synaptic Plasticity

PubMed Central

Clarke, Gwenaëlle L.; Chen, Jie; Nishimune, Hiroshi

2012-01-01

Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density) during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs), the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS), active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated. PMID:22438837

Direct Chronic Effect of Steroid Hormones in Attenuating Uterine Arterial Myogenic Tone: Role of PKC/ERK1/2

PubMed Central

Xiao, Daliao; Huang, Xiaohui; Yang, Shumei; Zhang, Lubo

2009-01-01

Pregnancy is associated with a significant decrease in uterine vascular tone and an increase in uterine blood flow. The present study tested the hypothesis that estrogen and progesterone differentially regulate the ERK1/2 and PKC signaling pathways in vascular smooth muscle resulting in a decrease in uterine vascular myogenic tone in pregnancy. Uterine arteries were isolated from nonpregnant (NPUA) and near-term pregnant (PUA) sheep. Chronic treatment (48 h) of NPUA with 17β-estradiol and progesterone caused a significant decrease in PKC-mediated contractions and pressure-induced myogenic tone. In accordance, treatment of PUA for 48 h with ICI 182,780 and RU 486 significantly increased PKC-induced contractions and myogenic tone. In contrast, acute treatment for 30 min had no effects on uterine artery contractility. An ERK1/2 inhibitor PD098059 restored the chronic effect of steroids on PKC-mediated contractions in NPUA. ERK1/2 protein and mRNA levels were greater in PUA as compared with NPUA. 17β-Estradiol and progesterone increased ERK1/2 protein in NPUA. In agreement, ICI 182,780 and RU 486 caused a significant decrease in ERK1/2 protein in PUA. Western blot showed six PKC isozymes, α, βI, βII, δ, ε and ζ in the uterine arteries. 17β-Estradiol and progesterone decreased the particulate-to-cytosolic ratio of PKCα, ε, and ζ, respectively, in NPUA. ICI 182,780 and RU 486 increased them in PUA. The results indicate a direct chronic effect of the steroid hormones in the up-regulation of ERK1/2 expression and down-regulation of PKC signaling pathway, resulting in attenuated myogenic tone of uterine artery in pregnancy. PMID:19528364

The Adequate Corpus Luteum: miR-96 Promotes Luteal Cell Survival and Progesterone Production.

PubMed

Mohammed, Bushra T; Sontakke, Sadanand D; Ioannidis, Jason; Duncan, W Colin; Donadeu, F Xavier

2017-07-01

Inadequate progesterone production from the corpus luteum is associated with pregnancy loss. Data available in model species suggest important roles of microRNAs (miRNAs) in luteal development and maintenance. To comprehensively investigate the involvement of miRNAs during the ovarian follicle-luteal transition. The effects of specific miRNAs on survival and steroid production by human luteinized granulosa cells (hLGCs) were tested using specific miRNA inhibitors. Candidate miRNAs were identified through microarray analyses of follicular and luteal tissues in a bovine model. An academic institution in the United Kingdom associated with a teaching hospital. hLGCs were obtained by standard transvaginal follicular-fluid aspiration from 35 women undergoing assisted conception. Inhibition of candidate miRNAs in vitro. Levels of miRNAs, mRNAs, FOXO1 protein, apoptosis, and steroids were measured in tissues and/or cultured cells. Two specific miRNA clusters, miR-183-96-182 and miR-212-132, were dramatically increased in luteal relative to follicular tissues. miR-96 and miR-132 were the most upregulated miRNAs within each cluster. Database analyses identified FOXO1 as a putative target of both these miRNAs. In cultured hLGCs, inhibition of miR-96 increased apoptosis and FOXO1 protein levels, and decreased progesterone production. These effects were prevented by small interfering RNA-mediated downregulation of FOXO1. In bovine luteal cells, miR-96 inhibition also led to increases in apoptosis and FOXO1 protein levels. miR-96 targets FOXO1 to regulate luteal development through effects on cell survival and steroid production. The miR-183-96-182 cluster could provide a novel target for the manipulation of luteal function. Copyright © 2017 Endocrine Society

Sex steroid modulation of cortisol secretion in sheep.

PubMed

van Lier, E; Carriquiry, M; Meikle, A

2014-06-01

There is strong evidence that the gonads modulate the hypothalamic-pituitary-adrenal axis. To investigate these sex differences at the adrenal glands of sheep we compared the cortisol response to ACTH (experiment 1) and measured the relative expression of oestrogen receptor alpha (ERS1), androgen receptor (AR), melanocortin 2 receptor (MC2R) and steroid acute regulatory protein (STAR) mRNA in adrenal glands (experiment 2) of gonadectomised rams and ewes either with or without sex steroid replacement. In experiment 1 six castrated adult rams and four ovariectomised adult ewes were used in two ACTH trials. On each trial blood samples were taken every 15 min for 4 h through an indwelling jugular catheter and each animal received 0.5 mg of an ACTH analogue i.v., immediately after the sample at 1 h from the beginning of the trial. Four days after the first trial the males received 100 mg of Testosterone Cyclopentilpropionate (TC) i.m. and the females received 2.5 mg of Oestradiol Benzoate (EB) i.m. At 72 h after TC or EB administration the second trial was performed. In experiment 2 the adrenal glands were obtained from gonadectomised adult rams (n=8) and adult ewes (n=8). Four rams received 100 mg of TC i.m. and four females received 0.5 mg of EB i.m. Blood samples were taken at 0, 12, 24, 48 and 72 h relative to steroid replacement and the animals were thereafter slaughtered. Cortisol, testosterone and 17β-oestradiol were determined by radioimmunoanalysis. The transcripts of ERS1, AR, MC2R and STAR were determined by real-time reverse transcription PCR in adrenal tissue. Cortisol secretion was higher in female sheep than in male sheep, and higher in EB-treated than non-treated ewes. No difference in cortisol secretion was observed between TC-treated and non-treated rams. Gonadectomised rams treated with TC presented greater AR mRNA and MC2R mRNA expression than males without the steroid replacement. Gonadectomised ewes treated with EB tended to present lower AR mRNA than the ones without steroid replacement. Gonadectomised rams with TC also had greater AR mRNA, ERS1 mRNA and MC2R mRNA expression than ewes treated with EB. The relative amount of STAR transcript was not different among the different groups. The results confirm sex differences in ACTH-induced cortisol secretion in sheep, as well as in the expression of the receptor proteins for both 17β-oestradiol and testosterone in the sheep adrenal gland. However, the underlying mechanisms for sex steroid modulation remain unresolved.

Altered adrenal steroid metabolism underlying hypercortisolism in female endurance athletes.

PubMed

Lindholm, C; Hirschberg, A L; Carlström, K; von Schoultz, B

1995-06-01

To explore possible changes in adrenal steroid metabolism and androgenic-anabolic status in female endurance athletes as a mechanism for their hypercortisolism. Adrenal steroids and androgenic-anabolic factors were studied during basal conditions and in response to ACTH stimulation related to menstrual status. Department of Obstetrics and Gynecology, Karolinska Hospital, Stockholm, Sweden. Thirteen female elite middle to long distance runners (six eumenorrheic, seven oligoamenorrheic) and seven regularly menstruating controls. Blood samples were collected before and after an injection of 250 micrograms IV synthetic ACTH 1-24. Body weight, height, and body fat were measured. Basal serum concentrations of cortisol, androstenedione (A), DHEA, DHEAS, 17 alpha-hydroxyprogesterone (17-OHP), T, steroid-binding proteins, and insulin-like growth factor I and ACTH-induced response (area under the curve) of cortisol, DHEA, and 17-OHP. Oligoamenorrheic athletes had higher basal cortisol and A concentrations compared with healthy controls, whereas basal levels of DHEA and DHEAS were normal. Important findings in the oligoamenorrheic athletes were a significantly lower ratio between the ACTH-induced increments of DHEA and 17-OHP and an increased ratio between basal A and DHEAS. Insulin-like growth factor I was correlated negatively to sex hormone-binding globulin and to the amount of body fat in the combined material. The results indicate a redistribution of adrenal steroid metabolism in favor of glucocorticoid production in female endurance athletes. We suggest that hypercortisolism in female endurance athletes is a physiological adaptation to maintain adequate blood glucose levels during a condition of energy deficiency.

ATF2 impairs glucocorticoid receptor–mediated transactivation in human CD8+ T cells

PubMed Central

Li, Ling-bo; Leung, Donald Y. M.; Strand, Matthew J.

2007-01-01

Chronic inflammatory diseases often have residual CD8+ T-cell infiltration despite treatment with systemic corticosteroids, which suggests divergent steroid responses between CD4+ and CD8+ cells. To examine steroid sensitivity, dexamethasone (DEX)–induced histone H4 lysine 5 (K5) acetylation and glucocorticoid receptor α (GCRα) translocation were evaluated. DEX treatment for 6 hours significantly induced histone H4 K5 acetylation in normal CD4+ cells (P = .001) but not in CD8+ cells. DEX responses were functionally impaired in CD8+ compared with CD4+ cells when using mitogen-activated protein kinase phosphatase (1 hour; P = .02) and interleukin 10 mRNA (24 hours; P = .004) induction as a readout of steroid-induced transactivation. Normal DEX-induced GCRα nuclear translocation and no significant difference in GCRα and GCRβ mRNA expression were observed in both T-cell types. In addition, no significant difference in SRC-1, p300, or TIP60 expression was found. However, activating transcription factor-2 (ATF2) expression was significantly lower in CD8+ compared with CD4+ cells (P = .009). Importantly, inhibition of ATF2 expression by small interfering RNA in CD4+ cells resulted in inhibition of DEX-induced transactivation in CD4+ cells. The data indicate refractory steroid-induced transactivation but similar steroid-induced transrepression of CD8+ cells compared with CD4+ cells caused by decreased levels of the histone acetyltransferase ATF2. PMID:17525285

Nandrolone, an anabolic steroid, stabilizes Numb protein through inhibition of mdm2 in C2C12 myoblasts.

PubMed

Liu, Xin-Hua; Yao, Shen; Levine, Alice C; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Collier, Lauren; Bauman, William A; Cardozo, Christopher P

2012-01-01

Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.

Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites

PubMed Central

2014-01-01

Background Steroids are lipophilic compounds with a gonane skeleton and play an important role in higher organisms. Due to different functionalizations - mainly hydroxylations - at the steroid molecule, they vary highly in their mode of action. The pharmaceutical industry is, therefore, interested in hydroxysteroids as therapeutic agents. The insertion of hydroxyl groups into a steroid core, however, is hardly accomplishable by classical chemical means; that is because microbial steroid hydroxylations are investigated and applied since decades. CYP106A2 is a cytochrome P450 monooxygenase from Bacillus megaterium ATCC 13368, which was first described in the late 1970s and which is capable to hydroxylate a variety of 3-oxo-delta4 steroids at position 15beta. CYP106A2 is a soluble protein, easy to express and to purify in high amounts, which makes this enzyme an interesting target for biotechnological purposes. Results In this work a focused steroid library was screened in vitro for new CYP106A2 substrates using a reconstituted enzyme assay. Five new substrates were identified, including dehydroepiandrosterone and pregnenolone. NMR-spectroscopy revealed that both steroids are mainly hydroxylated at position 7beta. In order to establish a biotechnological system for the preparative scale production of 7beta-hydroxylated dehydroepiandrosterone, whole-cell conversions with growing and resting cells of B. megaterium ATCC1336 the native host of CYP1062 and also with resting cells of a recombinant B. megaterium MS941 strain overexpressing CYP106A2 have been conducted and conversion rates of 400 muM/h (115 mg/l/h) were obtained. Using the B. megaterium MS941 overexpression strain, the selectivity of the reaction was improved from 0.7 to 0.9 for 7beta-OH-DHEA. Conclusions In this work we describe CYP106A2 for the first time as a regio-selective hydroxylase for 3-hydroxy-delta5 steroids. DHEA was shown to be converted to 7beta-OH-DHEA which is a highly interesting human metabolite, supposed to act as neuroprotective, anti-inflammatory and immune-modulatory agent. Optimization of the whole-cell system using different B. megaterium strains lead to a conversion of DHEA with B. megaterium showing high selectivity and conversion rates and displaying a volumetric yield of 103 mg/l/h 7beta-OH-DHEA. PMID:24903845

Steroid conversion with CYP106A2 - production of pharmaceutically interesting DHEA metabolites.

PubMed

Schmitz, Daniela; Zapp, Josef; Bernhardt, Rita

2014-06-05

Steroids are lipophilic compounds with a gonane skeleton and play an important role in higher organisms. Due to different functionalizations - mainly hydroxylations - at the steroid molecule, they vary highly in their mode of action. The pharmaceutical industry is, therefore, interested in hydroxysteroids as therapeutic agents. The insertion of hydroxyl groups into a steroid core, however, is hardly accomplishable by classical chemical means; that is because microbial steroid hydroxylations are investigated and applied since decades. CYP106A2 is a cytochrome P450 monooxygenase from Bacillus megaterium ATCC 13368, which was first described in the late 1970s and which is capable to hydroxylate a variety of 3-oxo-delta4 steroids at position 15beta. CYP106A2 is a soluble protein, easy to express and to purify in high amounts, which makes this enzyme an interesting target for biotechnological purposes. In this work a focused steroid library was screened in vitro for new CYP106A2 substrates using a reconstituted enzyme assay. Five new substrates were identified, including dehydroepiandrosterone and pregnenolone. NMR-spectroscopy revealed that both steroids are mainly hydroxylated at position 7beta. In order to establish a biotechnological system for the preparative scale production of 7beta-hydroxylated dehydroepiandrosterone, whole-cell conversions with growing and resting cells of B. megaterium ATCC1336 the native host of CYP1062 and also with resting cells of a recombinant B. megaterium MS941 strain overexpressing CYP106A2 have been conducted and conversion rates of 400 muM/h (115 mg/l/h) were obtained. Using the B. megaterium MS941 overexpression strain, the selectivity of the reaction was improved from 0.7 to 0.9 for 7beta-OH-DHEA. In this work we describe CYP106A2 for the first time as a regio-selective hydroxylase for 3-hydroxy-delta5 steroids. DHEA was shown to be converted to 7beta-OH-DHEA which is a highly interesting human metabolite, supposed to act as neuroprotective, anti-inflammatory and immune-modulatory agent. Optimization of the whole-cell system using different B. megaterium strains lead to a conversion of DHEA with B. megaterium showing high selectivity and conversion rates and displaying a volumetric yield of 103 mg/l/h 7beta-OH-DHEA.

The Tomato Hybrid Proline-Rich Protein regulates the abcission zone competence to respond to ethylene signals

USDA-ARS?s Scientific Manuscript database

The Tomato Hybrid Proline-Rich Protein (THyPRP) gene was specifically expressed in the tomato (Solanum lycopersicum) flower abscission zone (FAZ), and its stable antisense silencing under the control of an abscission zone (AZ)-specific promoter, Tomato Abscission Polygalacturonase4,significantly inh...

Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors

PubMed Central

Natale, Christopher A; Duperret, Elizabeth K; Zhang, Junqian; Sadeghi, Rochelle; Dahal, Ankit; O'Brien, Kevin Tyler; Cookson, Rosa; Winkler, Jeffrey D; Ridky, Todd W

2016-01-01

The association between pregnancy and altered cutaneous pigmentation has been documented for over two millennia, suggesting that sex hormones play a role in regulating epidermal melanocyte (MC) homeostasis. Here we show that physiologic estrogen (17β-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that normal primary human MCs lack classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G protein-coupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics. DOI: http://dx.doi.org/10.7554/eLife.15104.001 PMID:27115344

The Dynamics of Neurosteroids and Sex-Related Hormones in the Pathogenesis of Alzheimer's Disease.

PubMed

Hasanpour, Milad; Nourazarian, Alireza; Geranmayeh, Mohammad Hossein; Nikanfar, Masoud; Khaki-Khatibi, Fatemeh; Rahbarghazi, Reza

2018-05-04

Alzheimer's disease (AD) is commonly diagnosed by vast extracellular amyloid deposits and existence of intracellular neurofibrillary tangles. In accordance with the literature, age-related loss of sex steroid hormones in either males or females was found in relation to AD subjects. The dynamics of these hormones have been previously described in both physiological and pathological conditions with the evidence of changes in various intracellular signalings regarding the neurodegenerative disease. The potent protective effects of sex steroid hormones and their synthetic analogs are indicative of the decrease in the accumulated levels of intercellular beta-amyloid (Aβ) protein and an increase of specific proteases activity, resulting in the improvement of pathological features. In the current review, we focused on the dynamic of signaling pathway related to sex steroid hormones. It is logical to hypothesize that androgen hormones have regulatory actions on the kinetics of Aβ which make them as a promising preventive approach for neurodegenerative diseases in the near future.

Multi-organ damage induced by anabolic steroid supplements: a case report and literature review

PubMed Central

Samaha, Ali A; Nasser-Eddine, Walid; Shatila, Elizabeth; Haddad, John J; Wazne, Jaafar; Eid, Ali H

2008-01-01

Introduction The use of anabolic supplements and other related drugs for body building and to enhance athletic performance is nowadays widespread and acutely pervasive all around the world. This alarming increase in the use of anabolic and amino acid supplements has been linked to a diverse array of pathologies. As previously reported, the abuse of androgenic steroids is not without severe physiological, psychiatric and physical costs. The case we report here describes multi-organ damage resulting from the abuse and uncontrolled use of anabolic steroid supplements, mainly testosterone. Case presentation A 24-year-old white man presented with abdominal pain concomitant with nausea and vomiting. Laboratory analysis revealed hypercalcemia, elevated liver enzymes and high levels of amylase, lipase and creatine protein kinase. Conclusion Amino acid as well as anabolic supplements may lead to abnormal functioning of many organs, which could be fatal in some instances. This mandates worldwide and concerted efforts to educate the public, especially the youth, about the dangers of these increasingly abused drugs. PMID:18976461

Androgens and the male reproductive tract: an overview of classical roles and current perspectives.

PubMed

Patrão, Marilia T C C; Silva, Erick J R; Avellar, Maria Christina W

2009-11-01

Androgens are steroid hormones that play key roles in the development and maintenance of male phenotype and reproductive function. These hormones also affect the function of several non-reproductive organs, such as bone and skeletal muscle. Endogenous androgens exert most of their effects by genomic mechanisms, which involve hormone binding to the androgen receptor (AR), a ligand-activated transcription factor, resulting in the modulation of gene expression. AR-induced non-genomic mechanisms have also been reported. A large number of steroidal and non-steroidal AR-ligands have been developed for therapeutic use, including the treatment of male hypogonadism (AR agonists) and prostate diseases (AR antagonists), among other pathological conditions. Here, the AR gene and protein structure, mechanism of action and AR gene homologous regulation were reviewed. The AR expression pattern, its in vivo regulation and physiological relevance in the developing and adult testis and epididymis, which are sites of sperm production and maturation, respectively, were also presented.

Sexually dimorphic expression of the genes encoding ribosomal proteins L17 and L37 in the song control nuclei of juvenile zebra finches

PubMed Central

Tang, Yu Ping; Wade, Juli

2010-01-01

Studies evaluating the role of steroid hormones in sexual differentiation of the zebra finch song system have produced complicated and at times paradoxical results, and indicate that additional factors may be critical. Therefore, in a previous study we initiated a screen for differential gene expression in the telencephalon of developing male and female zebra finches. The use of cDNA microarrays and real-time quantitative PCR revealed increased expression of the genes encoding ribosomal proteins L17 and L37 (RPL17 and RPL37) in the male forebrain as a whole. Preliminary in situ hybridization data then indicated enhanced expression of both these genes in song control regions. Two experiments in the present study quantified the mRNA expression. The first utilized 25-day-old male and female zebra finches. The second compared a separate set of juveniles to adults of both sexes to both re-confirm enhanced expression in juvenile males and to determine whether it is limited to developing animals. In Experiment 1, males exhibited increased expression of both RPL17 and RPL37 compared to females in Area X, the robust nucleus of the arcopallium (RA), and the ventral ventricular zone (VVZ), which may provide neurons to Area X. Experiment 2 replicated the sexually dimorphic expression of these genes at post-hatching day 25, and documented that the sex differences are eliminated or greatly reduced in adults. The results are consistent with the idea that these ribosomal proteins may influence sexual differentiation of Area X and RA, potentially regulating the genesis and/or survival of neurons. PMID:16938280

Sexually dimorphic expression of the genes encoding ribosomal proteins L17 and L37 in the song control nuclei of juvenile zebra finches.

PubMed

Tang, Yu Ping; Wade, Juli

2006-12-18

Studies evaluating the role of steroid hormones in sexual differentiation of the zebra finch song system have produced complicated and at times paradoxical results, and indicate that additional factors may be critical. Therefore, in a previous study we initiated a screen for differential gene expression in the telencephalon of developing male and female zebra finches. The use of cDNA microarrays and real-time quantitative PCR revealed increased expression of the genes encoding ribosomal proteins L17 and L37 (RPL17 and RPL37) in the male forebrain as a whole. Preliminary in situ hybridization data then indicated enhanced expression of both these genes in song control regions. Two experiments in the present study quantified the mRNA expression. The first utilized 25-day-old male and female zebra finches. The second compared a separate set of juveniles to adults of both sexes to both re-confirm enhanced expression in juvenile males and to determine whether it is limited to developing animals. In Experiment 1, males exhibited increased expression of both RPL17 and RPL37 compared to females in Area X, the robust nucleus of the arcopallium (RA), and the ventral ventricular zone (VVZ), which may provide neurons to Area X. Experiment 2 replicated the sexually dimorphic expression of these genes at post-hatching day 25, and documented that the sex differences are eliminated or greatly reduced in adults. The results are consistent with the idea that these ribosomal proteins may influence sexual differentiation of Area X and RA, potentially regulating the genesis and/or survival of neurons.

Rat prostatic steroid binding protein: DNA sequence and transcript maps of the two C3 genes.

PubMed Central

Hurst, H C; Parker, M G

1983-01-01

In the rat there are two non-allelic genes C3(1) and C3(2) for the C3 polypeptide of prostatic steroid binding protein. We have cloned and sequenced both genes and show that only C3(1) is responsible for the production of authentic C3. Although there is a marked difference in their transcriptional activity, the two genes share extensive DNA sequence homology there being only one base difference from nucleotide - 235 to within the first intron. Transcript mapping has shown that there are two distinct C3 transcripts which share a unique 3' terminus but have 5' termini 38 bases apart each preceded by a 'TATA' box homology. Interestingly, an identical repetitive element is present just upstream of both genes. Both families of transcripts, which are produced in a ratio of 18:1, are coordinately regulated by testosterone. Images Fig. 3. Fig. 4. Fig. 5. PMID:6685625

Binding of alkylphenols and alkylated non-phenolics to the rainbow trout (Oncorhynchus mykiss) plasma sex steroid-binding protein.

PubMed

Tollefsen, K-E

2007-09-01

Alkylphenols are well-known endocrine disrupters, mediating effects through the estrogen receptor (ER). Although the estrogenic properties of the alkylphenols are well documented, alternative mechanisms of action are poorly described. In the present work, the interaction of a range of alkyl-substituted phenols and alkyl-substituted non-phenolics with the rainbow trout (Oncorhynchus mykiss) sex steroid-binding protein (rtSBP) were determined by competitive ligand-binding studies. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure were assessed. The results showed that the rtSBP binds to most chemical structures tested, although the highest affinity was obtained for mono-substituted alkylphenols with a chain length of four to eight methyl groups. Interestingly, rtSBP binding was also observed for non-phenolic compounds such as 4-t-butylcyclohexanol and 4-t-butylnitrobenzene suggesting that the rtSBP has a broad binding specificity for alkylphenols and alkylated non-phenolics.

Effects of androgen-binding protein (ABP) on spermatid Tnp1 gene expression in vitro.

PubMed

Della-Maria, Julie; Gerard, Anne; Franck, Patricia; Gerard, Hubert

2002-12-30

In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells. Copyright 2002 Elsevier Science Ireland Ltd.

High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).

PubMed

Sluchanko, Nikolai N; Tugaeva, Kristina V; Faletrov, Yaroslav V; Levitsky, Dmitrii I

2016-03-01

Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production. Copyright © 2015 Elsevier Inc. All rights reserved.

Relation of uptake and metabolism of (1,2,6,7-3H)testosterone to individual differences in sexual behavior in male guinea pigs.

PubMed

Harding, C F; Feder, H H

1976-03-19

Male guinea pigs were given 3 tests for sexual behavior. Animals that never ejaculated were classified as low activity (LA), animals that ejaculated on one test were classified as medium activity (MA), and animals that ejaculated on two or more tests were classified as high activity (HA). Subsequently, animals from each group were castrated and given an s.c. injection of 43 muCi of [1,2,6,7-3H]testosterone and were killed 0.5, 1, or 4 h after injection. There were no significant differences in uptake or metabolism of radioactive testosterone among LA, MA, and HA males in homogenates of anterior and posterior hypothalamus, cerebral cortex, midbrain, or seminal vesicle. Thus, differences in sexual behavior could not be attributed to differences in testosterone uptake in tissue homogenates. At the 1 h time interval (time of peak plasma radioactivity), radioactivity in the seminal vesicles of all males was primarily in the form of steroids with the chromatographic mobility of dihydrotestosterone. In all males, anterior and posterior hypothalamus contained a higher proportion of steroids with the mobility of testosterone than did midbrain, and midbrain contained more testosterone zone radioactivity than cerebral cortex at 1 h. The highest proportion of dihydrotestosterone zone radioactivity in neural tissues was found in anterior hypothalamus. These results are discussed in terms of androgenic mediation of sex behavior by the anterior hypothalamus in guinea pigs.

Protein expression differs between neural progenitor cells from the adult rat brain subventricular zone and olfactory bulb.

PubMed

Maurer, Martin H; Feldmann, Robert E; Bürgers, Heinrich F; Kuschinsky, Wolfgang

2008-01-16

Neural progenitor cells can be isolated from various regions of the adult mammalian brain, including the forebrain structures of the subventricular zone and the olfactory bulb. Currently it is unknown whether functional differences in these progenitor cell populations can already be found on the molecular level. Therefore, we compared protein expression profiles between progenitor cells isolated from the subventricular zone and the olfactory bulb using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry. The subventricular zone and the olfactory bulb are connected by the Rostral Migratory Stream (RMS), in which glial fibrillary acidic protein (GFAP)-positive cells guide neuroblasts. Recent literature suggested that these GFAP-positive cells possess neurogenic potential themselves. In the current study, we therefore compared the cultured neurospheres for the fraction of GFAP-positive cells and their morphology of over a prolonged period of time. We found significant differences in the protein expression patterns between subventricular zone and olfactory bulb neural progenitor cells. Of the differentially expressed protein spots, 105 were exclusively expressed in the subventricular zone, 23 showed a lower expression and 51 a higher expression in the olfactory bulb. The proteomic data showed that more proteins are differentially expressed in olfactory bulb progenitors with regard to proteins involved in differentiation and microenvironmental integration, as compared to the subventricular zone progenitors. Compared to 94% of all progenitors of the subventricular zone expressed GFAP, nearly none in the olfactory bulb cultures expressed GFAP. Both GFAP-positive subpopulations differed also in morphology, with the olfactory bulb cells showing more branching. No differences in growth characteristics such as doubling time, and passage lengths could be found over 26 consecutive passages in the two cultures. In this study, we describe differences in protein expression of neural progenitor populations isolated from two forebrain regions, the subventricular zone and the olfactory bulb. These subpopulations can be characterized by differential expression of marker proteins. We isolated fractions of progenitor cells with GFAP expression from both regions, but the GFAP-positive cells differed in number and morphology. Whereas in vitro growth characteristics of neural progenitors are preserved in both regions, our proteomic and immunohistochemical data suggest that progenitor cells from the two regions differ in morphology and functionality, but not in their proliferative capacity.

Culture of prostate epithelial cells of the rhesus monkey on extracellular matrix substrate: influence of steroids and insulin-like growth factors.

PubMed

Udayakumar, T S; Jeyaraj, D A; Rajalakshmi, M; Sharma, R S

1999-09-01

Rhesus monkey prostate epithelial cells from the cranial lobe were isolated and cultured in flasks coated either with collagen IV or laminin. The effects of stromal cell medium, androgens and growth factors on cell number, thymidine incorporation and secretory activity were assessed. The results indicate that dihydrotestosterone (DHT) and androstenedione have stimulatory influences on cell proliferation and secretion in coated flasks. DHT was more effective in increasing cell number but the induction of secretory activity was similar with both steroids. The combination of IGF-I and -II resulted in inducing better cell proliferation and secretory activity than the individual IGFs but, of the two IGFs, IGF-I was more effective than IGF-II. DHT with IGFs was more potent in inducing proliferation, differentiation and secretion than androstenedione. Even in the absence of steroids or growth factors, colony formation and confluence occurred in coated flasks but cell differentiation and secretion only to a limited extent. In conclusion, we were able to establish an in vitro primary culture of prostate epithelial cells from rhesus monkey using extracellular matrix proteins, steroids and growth factors as additional supplements. This culture system may be useful to study prostate cell physiology and to identify drugs that can inhibit cell proliferation.

Anabolic androgenic steroid nandrolone decanoate reduces hypothalamic proopiomelanocortin mRNA levels.

PubMed

Lindblom, Jonas; Kindlundh, Anna M S; Nyberg, Fred; Bergström, Lena; Wikberg, Jarl E S

2003-10-03

Supratherapeutical doses of anabolic androgenic steroids (AASs) have dramatic effects on metabolism in humans, and also inhibit feeding and reduce the rate of body weight gain in rats. In order to test the hypothesis that the AAS metabolic syndrome is accompanied by alterations in the central melanocortin system, we evaluated body weight, food intake and hypothalamic agouti-related protein (AgRP) and proopiomelanocortin (POMC) mRNA levels following administration of different doses of the anabolic androgenic steroid nandrolone decanoate. In order to distinguish changes induced by the steroid treatment per se from those resulting from the reduced food intake and growth rate, we also compared the effect of nandrolone decanoate on AgRP and POMC mRNA expression with both normally fed, and food restricted control groups. We here report that administration of nandrolone specifically reduces arcuate nucleus POMC mRNA levels while not affecting the expression level of AgRP. The effect on POMC expression was not observed in the food restricted controls, excluding the possibility that the observed effect was a mere response to the reduced food intake and body weight. These results raise the possibility that some of the metabolic and behavioural consequences of AAS abuse may be the result of alterations in the melanocortin system.

Sex Steroid Modulation of Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

PubMed Central

Ockner, Robert K.; Lysenko, Nina; Manning, Joan A.; Monroe, Scott E.; Burnett, David A.

1980-01-01

The mechanism by which sex steroids influence very low density hepatic lipoprotein triglyceride production has not been fully elucidated. In previous studies we showed that [14C]oleate utilization and incorporation into triglycerides were greater in hepatocyte suspensions from adult female rats than from males. The sex differences were not related to activities of the enzymes of triglyceride biosynthesis, whereas fatty acid binding protein (FABP) concentration in liver cytosol was greater in females. These findings suggested that sex differences in lipoprotein could reflect a sex steroid influence on the availability of fatty acids for hepatocellular triglyceride biosynthesis. In the present studies, sex steroid effects on hepatocyte [14C]oleate utilization and FABP concentration were investigated directly. Hepatocytes from immature (30-d-old) rats exhibited no sex differences in [14C]oleate utilization. With maturation, total [14C]oleate utilization and triglyceride biosynthesis increased moderately in female cells and decreased markedly in male cells; the profound sex differences in adults were maximal by age 60 d. Fatty acid oxidation was little affected. Rats were castrated at age 30 d, and received estradiol, testosterone, or no hormone until age 60 d, when hepatocyte [14C]oleate utilization was studied. Castration virtually eliminated maturational changes and blunted the sex differences in adults. Estradiol or testosterone largely reproduced the appropriate adult pattern of [14C]oleate utilization regardless of the genotypic sex of the treated animal. In immature females and males, total cytosolic FABP concentrations were similar. In 60-d-old animals, there was a striking correlation among all groups (females, males, castrates, and hormone-treated) between mean cytosolic FABP concentration on the one hand, and mean total [14C]oleate utilization (r = 0.91) and incorporation into triglycerides (r = 0.94) on the other. In 30-d-old animals rates of [14C]oleate utilization were greater, relative to FABP concentrations, than in 60-d-old animals. The sex differences that characterize fatty acid utilization in adult rat hepatocytes are not present in cells from immature animals, and reflect in part the influence of sex steroids. It remains to be determined whether the observed relationship of hepatic FABP concentration to [14C]oleate utilization in adult cells is causal or secondary to changes in cellular fatty acid uptake effected through another mechanism. In either case, modulation of triglyceride-rich lipoprotein production by six steroids appears to be mediated to a significant extent by their effects on hepatic fatty acid utilization. PMID:7364935

Recent Advances in Drug Design and Drug Discovery for Androgen- Dependent Diseases.

PubMed

Cabeza, Marisa; Sánchez-Márquez, Araceli; Garrido, Mariana; Silva, Aylín; Bratoeff, Eugene

2016-01-01

This article summarizes the importance of different targets such as 5α-reductase, 17β-HSD, CYP17A, androgen receptor and protein kinase A for the treatment of prostate cancer and benign prostatic hyperplasia. It is a well known fact that dihydrotestosterone (DHT) is associated with the development of androgen-dependent afflictions. At the present time, several research groups are attempting to develop new steroidal and non-steroidal molecules with the purpose of inhibiting the synthesis and biological response of DHT. This review also discusses the most recent studies reported in the literature that describe the therapeutic potential of novel compounds, as well as the new drugs, principally inhibitors of 5α-reductase.

Chemical constituents of the genus Polygonatum and their role in medicinal treatment.

PubMed

Zhao, Xueying; Li, Ji

2015-04-01

Polygonatum is a famous traditional Chinese medicine that is widely used in China, Korea and Japan. In the last decade, constituents of the genus have been reported including steroidal saponins, flavones, alkaloids, lignins, amino acids and carbohydrates, some of which show biological properties such as antiviral and antitumor activity, variable effects on the immune system and anticoagulant activity. In addition, some findings provide novel evidence that Polygonatum species may contain potential anti-tumor and anti-viral proteins for possible medical application and large-scale pharmaceutical production. In this review, we focus on the updated research of the chemical constituents of Polygonatum including polysaccharides, steroidal saponins, flavonoids and lectins, and their potential therapeutic roles.

High protein buckwheat flour suppresses hypercholesterolemia in rats and gallstone formation in mice by hypercholesterolemic diet and body fat in rats because of its low protein digestibility.

PubMed

Tomotake, Hiroyuki; Yamamoto, Naoe; Yanaka, Noriyuki; Ohinata, Hiroshi; Yamazaki, Rikio; Kayashita, Jun; Kato, Norihisa

2006-02-01

This study evaluated the physiologic properties of high protein buckwheat flour (PBF) by examining its effects on serum cholesterol and body fat in rats and on cholesterol gallstone formation in mice. Animals were fed experimental diets that contained casein, buckwheat protein extract (BWP), or PBF as a protein source (net protein content 200 g/kg). In experiment 1, consumption of PBF and BWP for 10 d caused 33% and 31% decreases, respectively, in serum cholesterol of rats fed cholesterol-enriched diets when compared with consumption of casein (P < 0.05). Dietary PBF caused a significant decrease in liver cholesterol, whereas dietary BWP caused only a slight decrease (P > 0.05). Fecal excretion of neutral and acidic steroids in the PBF group was significantly higher than those in the BWP and casein groups. In experiment 2, consumption of PBF for 10 d significantly suppressed adipose tissue weight and hepatic activity of fatty acid synthase in rats fed cholesterol-free diets compared with consumption of casein (P < 0.05), whereas that of BWP for this period caused only a slight decrease in adipose tissue weight (P > 0.05). In experiment 3, dietary PBF and BWP significantly decreased the incidence of cholesterol gallstones and lithogenic index in mice fed cholesterol-enriched diets for 27 d, which was associated with increased fecal excretion of acidic steroids. This study demonstrated that PBF has strong activities against hypercholesterolemia, obesity, and gallstone formation, suggesting a potential usefulness of PBF as functional ingredient.

Estradiol, progesterone and genistein differentially regulate levels of aquaporin (AQP)-1, 2, 5 and 7 expression in the uteri of ovariectomized, sex-steroid deficient rats.

PubMed

Chinigarzadeh, Asma; Muniandy, Sekaran; Salleh, Naguib

2016-11-01

In this study, effects of estradiol, progesterone and genistein on uterine aquaporin (AQP)-1, 2, 5 and 7 expression were investigated in sex-steroid deficient state which could help to elucidate the mechanisms underlying uterine fluid volume changes that were reported under these hormone and hormone-like compound influences. Uteri from ovariectomized, female Sprague-Dawley rats receiving seven days estradiol, progesterone or genistein (25, 50 and 100mg/kg/day) were harvested and levels of AQP-1, 2, 5 and 7 proteins and mRNAs were determined by Western blotting and Real-time PCR (qPCR) respectively. Distribution of these proteins in uterus was observed by immunohistochemistry. Genistein caused a dose-dependent increase in uterine AQP-1, 2, 5 and 7 protein and mRNA expression, however at the levels lower than following estradiol or progesterone stimulations. Effects of genistein were antagonized by estradiol receptor blocker, ICI 182780. Estradiol caused the highest AQP-2 protein and mRNA expression while progesterone caused the highest AQP-1, 5 and 7 protein and mRNA expression in uterus. AQP-1, 2, 5 and 7 protein were found to be distributed in the myometrium as well as in uterine luminal and glandular epithelia and endometrial blood vessels. In conclusion, the observed effects of estradiol, progesterone and genistein on uterine AQP-1, 2, 5 and 7 expression could help to explain the differences in the amount of fluid accumulated in the uterus under these different conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

[Chromosomal proteins: histones and acid proteins].

PubMed

Salvini, M; Gabrielli, F

1976-01-01

Experimental data about the chemistry and the biology of chromosomal proteins are reviewed. Paragraphs include: aminoacid sequential data and post-translational covalent modications of histones, histone chemical differences in different tissues of the same species and in homologous organs of different species, histone synthesis subcellular localization and its association with DNA synthesis, histone synthesis transcriptional and translational control, histone synthesis during meiosis, oogenesis and early embryogenesis. The possible role of histones as controllers of gene expression is discussed and a model of primary structure of chromatine is proposed. The "acidic proteins" data concern the high tissue eterogenity of these proteins and their role in the steroid-hormon-controlled gene expression. The possible role of acidic proteins as general controllers of gene expression in eucariotic cells is discussed.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

PubMed

Bortfeld, M; Rius, M; König, J; Herold-Mende, C; Nies, A T; Keppler, D

2006-01-01

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

Pregnane Glycosides Interfere With Steroidogenic Enzymes to Down-Regulate Corticosteroid Production in Human Adrenocortical H295R Cells

PubMed Central

KOMARNYTSKY, SLAVKO; ESPOSITO, DEBORA; POULEV, ALEXANDER; RASKIN, ILYA

2013-01-01

A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias. In this study, we investigated the effects, structure-activity relationships, and mechanism of action of pregnane glycosides on steroidogenesis in human adrenocortical H295R cells. Administration of pregnane glycosides for 24 h suppressed the basal and forskolin-stimulated release of androstenedione, corticosterone, and cortisone from H295R cells. The conversion of progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to either androstenedione or 11-deoxycortisol was most strongly affected, with 12-cinnamoyl-, benzoyl-, and tigloyl-containing pregnanes showing the highest activity. Incubation of pregnane glycosides for 24 h had no effect on mRNA transcripts of CYP11A1, CYP21A1, CYP11B1 cytochrome enzymes and steroidogenic acute regulatory protein (StaR) protein, yet resulted in twofold decrease in HSD3B1 mRNA levels. At the same time, pregnane glycosides had no effect on the CYP1, 2, or 3 drug and steroid metabolism enzymes and showed weak Na+/K+ ATPase and glucocorticoid receptor binding. Taken together, these data suggest that pregnane glycosides specifically suppress steroidogenesis through strong inhibition of 11β-hydroxylase and steroid 17-alpha-monooxygenase, and weak inhibition of cytochrome P450 side chain cleavage enzyme and 21β-hydroxylase, but not 3β-hydroxysteroid dehydrogenase/isomerase. PMID:23065845

Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster

PubMed Central

Kaieda, Yuya; Masuda, Ryota; Nishida, Ritsuo; Shimell, MaryJane; O’Connor, Michael B.; Ono, Hajime

2018-01-01

Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. We manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. We monitored expression of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, we found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. We suggest that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression. PMID:28782527

Implantation failure in mice with a disruption in Phospholipase C beta 1 gene: lack of embryonic attachment, aberrant steroid hormone signalling and defective endocannabinoid metabolism

PubMed Central

Filis, Panayiotis; Kind, Peter C.; Spears, Norah

2013-01-01

Phospholipase C beta 1 (PLCβ1) is a downstream effector of G-protein-coupled receptor signalling and holds central roles in reproductive physiology. Mice with a disruption in the Plcβ1 gene are infertile with pleiotropic reproductive defects, the major reproductive block in females being implantation failure. Here, PLCβ1 was demonstrated at the luminal and glandular epithelia throughout the pre- and peri-implantation period, with transient stromal expression during 0.5–1.5 days post coitum (dpc). Examination of implantation sites at 4.5 dpc showed that in females lacking functional PLCβ1 (knock-out (KO) females), embryos failed to establish proper contact with the uterine epithelium. Proliferating luminal epithelial cells were evident in KO implantation sites, indicating failure to establish a receptive uterus. Real-time PCR demonstrated that KO implantation sites had aberrant ovarian steroid signalling, with high levels of estrogen receptor α, lactoferrin and amphiregulin mRNA, while immunohistochemistry revealed very low levels of estrogen receptor α protein, possibly due to rapid receptor turnover. KO implantation sites expressed markedly less fatty acid amide hydrolase and monoacylglycerol lipase, indicating that endocannabinoid metabolism was also affected. Collectively, our results show that PLCβ1 is essential for uterine preparation for implantation, and that defective PLCβ1-mediated signalling during implantation is associated with aberrant ovarian steroid signalling and endocannabinoid metabolism. PMID:23295235

Steroidal constituents from the edible sea urchin Diadema savignyi Michelin induce apoptosis in human cancer cells.

PubMed

Thao, Nguyen Phuong; Luyen, Bui Thi Thuy; Kim, Eun Ji; Kang, Jung Il; Kang, Hee Kyoung; Cuong, Nguyen Xuan; Nam, Nguyen Hoai; Kiem, Phan Van; Minh, Chau Van; Kim, Young Ho

2015-01-01

Bioassay-directed fractionation and purification were used to isolate 12 steroids (1-12) from a CH(2)Cl(2) extract of the edible Vietnamese sea urchin Diadema savignyi Michelin. The cytotoxic activity of the CH(2)Cl(2) extract and 12 steroids was evaluated in three human cancer cell lines (HL-60, PC-3, and SNU-C5). Relative to the effects of the positive control, mitoxantrone, the CH(2)Cl(2) extract (with an inhibitory concentration of 50% [IC(50)] values ranging from 1.37±0.15 to 3.11±0.15 μg/mL) and compounds 2 (with IC(50) values ranging from 5.29±0.11 to 6.80±0.67 μM) and 11 (with IC(50) values ranging from 4.95±0.07 to 6.99±0.28 μM) exhibited potent cytotoxic effects against all three tested human cancer cell lines. In addition, the CH(2)Cl(2) extract and compounds 2 and 11 were found to induce apoptosis. The induction of apoptosis was accompanied by alterations of the apoptosis-related protein expression, inactivation of ERK1/2 mitogen-activated protein kinase signaling, and decreased c-Myc expression. These data suggest that compounds 2 and 11 from the edible sea urchin D. savignyi may have potential for the treatment of colon cancer, leukemia, and prostate cancer as complementary cancer remedies.

Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster.

PubMed

Kaieda, Yuya; Masuda, Ryota; Nishida, Ritsuo; Shimell, MaryJane; O'Connor, Michael B; Ono, Hajime

2017-10-01

Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. We manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. We monitored expression of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, we found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. We suggest that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression. Copyright © 2017 Elsevier Inc. All rights reserved.

Osteoporosis: Implications for Risk Reduction in the College Setting.

ERIC Educational Resources Information Center

Leslie, Maryann; St. Pierre, Richard W.

1999-01-01

Examines risk factors for osteoporosis that are especially relevant to the college health setting, focusing on bone development, inadequate calcium and vitamin D intake, cigarette smoking and alcohol use, steroid use and high protein diets, and physical inactivity and excessive exercise. Also presents intervention strategies for college health…

ALTERED SERUM SEX STEROIDS AND VITELLOGENIN INDUCTION IN WALLEYE (STIZOSTEDION VITREUM) COLLECTED NEAR A METROPOLITAN SEWAGE TREATMENT PLANT

EPA Science Inventory

Feral, male walleye collected from the Mississippi River below the St. Paul metropolitan sewage treatment plant (STP) contained measurable levels of the estrogen-inducible, female egg protein, vitellogenin. These same fish showed significantly decreased serum androgen and signifi...

INTERSPECIES EXTRAPOLATION AND IN VITRO SCREENING BASED UPON RECOMBINANT AR AND ER PROTEINS

EPA Science Inventory

While EDCs have the potential to act via several mechanisms of action, one of the most widely accepted is the ability of environmental chemicals to interact directly with either the estrogen (ER) or androgen receptor (AR). Although we know that the steroid receptors of the lower...

Endogenous sex steroids and cardio- and cerebro-vascular disease in the postmenopausal period.

PubMed

Pappa, Theodora; Alevizaki, Maria

2012-08-01

Cardio- and cerebro-vascular diseases are two leading causes of death and long-term disability in postmenopausal women. The acute fall of estrogen in menopause is associated with increased cardiovascular risk. The relative contribution of androgen to this risk is also being recognized. The use of more sensitive assays for estradiol measurement and the study of receptor and carrier protein gene polymorphisms have provided some new information on the clinical relevance of endogenous sex steroids. We provide an update on the role of endogenous sex steroids on cardio- and cerebro-vascular disease in the postmenopausal period. We performed a PubMed search using the terms 'endogenous estrogen', 'androgen', 'cardiovascular disease', 'cerebro-vascular disease', 'stroke', 'carotid artery disease', and 'subclinical atherosclerosis'. The majority of studies show a beneficial effect of endogenous estrogen on the vasculature; however, there are a few studies reporting the contrary. A significant body of literature has reported associations of endogenous estrogen and androgen with early markers of atherosclerosis and metabolic parameters. Data on the relevance of endogenous sex steroids in heart disease and stroke are inconclusive. Most studies support a beneficial role of endogenous estrogens and, probably, an adverse effect of androgens in the vasculature in postmenopausal women. However, the described associations may not always be considered as causal. It is possible that circulating estrogen might represent a marker of general health status or alternatively reflect the sum of endogenous androgens aromatized in the periphery. Elucidating the role of sex steroids in cardio- and cerebro-vascular disease remains an interesting field of future research.

Angiotensin II-aldosterone interaction in human coronary microarteries involves GPR30, EGFR, and endothelial NO synthase.

PubMed

Batenburg, Wendy W; Jansen, Pieter M; van den Bogaerdt, Antoon J; J Danser, Alexander H

2012-04-01

The aim of this study was to investigate the aldosterone-angiotensin (Ang) II interaction in human coronary microarteries (HCMAs). HCMAs, obtained from 75 heart-beating organ donors, were mounted in myographs and exposed to Ang II, either directly or following a 30-min pre-incubation with aldosterone, 17β-oestradiol, hydrocortisone, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, the extracellular regulated kinase 1/2 (ERK1/2) inhibitor PD98059, the GPR30 antagonist G15, or the epidermal growth factor receptor (EGFR) antagonist AG1478. Ang II constricted HCMAs in a concentration-dependent manner. All steroids, at nanomolar levels, potentiated Ang II and G15 prevented this effect. The potentiation disappeared or was reversed into Ang II antagonism at micromolar steroid levels. NO synthase (NOS) inhibition prevented the latter antagonism in the case of 17β-oestradiol, whereas both aldosterone and 17β-oestradiol at micro- (but not nano-) molar levels induced endothelial NOS phosphorylation in human umbilical vein endothelial cells. AG1478, but not SB203580 or PD98059, abolished the Ang II-induced contraction in the presence of aldosterone or 17β-oestradiol, and none of these drugs affected Ang II alone. Steroids including aldosterone affect Ang II-induced vasoconstriction in a biphasic manner. Potentiation occurs at nanomolar steroid levels and depends on GPR30 and EGFR transactivation. At micromolar steroid levels, this potentiation either disappears (aldosterone and hydrocortisone) or is reversed into an inhibition (17β-oestradiol), and this is due to the endothelial NOS activation that occurs at such concentrations.

Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Savechenkov, Pavel Y.; Chiara, David C.; Desai, Rooma

2017-08-01

Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but the locations of their GABAAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAAR, 11β-(4-azido-2,3,5,6-tetrafluorobenzoyloxy)allopregnanolone, (F4N3Bzoxy-AP) and 11-aziallopregnanolone (11-AziAP). Both photoprobes acted with equal or higher potency than alphaxalone as general anesthetics and potentiators of GABAAR responses, left-shifting the GABA concentration – response curve for human α1β3γ2 GABAARs expressed in Xenopus oocytes, and enhancing [3H]muscimol binding to α1β3γ2 GABAARs expressed in HEK293 cells. With EC50 of 110 nM, 11-AziAP is one the most potent general anestheticsmore » reported. [3H]F4N3Bzoxy-AP and [3H]11-AziAP, at anesthetic concentrations, photoincorporated into α- and β-subunits of purified α1β3γ2 GABAARs, but labeling at the subunit level was not inhibited by alphaxalone (30 μM). The enhancement of photolabeling by 3H-azietomidate and 3H-mTFD-MPAB in the presence of either of the two steroid photoprobes indicates the neurosteroid binding site is different from, but allosterically related to, the etomidate and barbiturate sites. Our observations are consistent with two hypotheses. First, F4N3Bzoxy-AP and 11-aziAP bind to a high affinity site in such a pose that the 11-photoactivatable moiety, that is rigidly attached to the steroid backbone, points away from the protein. Second, F4N3Bzoxy-AP, 11-aziAP and other steroid anesthetics, which are present at very high concentration at the lipid-protein interface due to their high lipophilicity, act via low affinity sites, as proposed by Akk et al. (Psychoneuroendocrinology 2009, 34S1, S59-S66).« less

The dipole moment of the electron carrier adrenodoxin is not critical for redox partner interaction and electron transfer.

PubMed

Hannemann, Frank; Guyot, Arnaud; Zöllner, Andy; Müller, Jürgen J; Heinemann, Udo; Bernhardt, Rita

2009-07-01

Dipole moments of proteins arise from helical dipoles, hydrogen bond networks and charged groups at the protein surface. High protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the electron transfer protein adrenodoxin and investigated the influence of this on protein-protein interactions and electron transfer. In bovine adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length adrenodoxin model based on the Adx(4-108) and the wild type adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this electron carrier the molecular dipole moment was systematically changed. Five recombinant adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner proteins and for their function during electron transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the protein function as compared with the unmodified adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the electron transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in electron transfer proteins is not involved in electrostatic steering among the proteins in the redox chain.

[Expression of ICAT and Wnt signaling-related proteins in the monocytic differentiation of HL-60 cells induced by a new steroidal drug NSC67657].

PubMed

Wang, J S; Wang, W J; Wang, T; Zhang, Y

2016-04-01

To investigate the expression of mRNA and proteins of β-catenin, TCF-4 (ICAT) and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new steroidal drug NSC67657. Wright's staining and α-NBE staining were used to observe the differentiation of HL-60 cells after 5 days of 10 μmol/L NSC67657 treatment. Flow cytometry (FCM) was used to detect the differentiation and cell cycles. The expressions of mRNA and proteins of ICAT and Wnt signaling pathway-related factors, including β-catenin, TCF-4, c-myc, cyclin D1 and TCF-1 before and after differentiation, were detected by RT-PCR and Western blot. Morphological observation showed that NSC67657 induced monocytic differentiation of HL-60 cells. At 5 days after 10 μmol/L NSC67657 treatment, the number of CD14(+) HL-60 cells was (94.37±2.84)%, significantly higher than the (1.31±0.09)% in control group (P<0.01). The flow cytometry assay revealed that NSC67657 induced (76.46±2.83)% of G1/G0 phase arrest, significantly higher than that of (59.40±5.42)% in the control group (P<0.05), while the S phase cells were of (18.76±0.98)%, significantly lower than that of (34.38±2.61) % in the control group (P<0.05). The NSC67657 treatment also up-regulated the expression of ICAT mRNA and protein, and down-regulated the expression of β-catenin mRNA and protin (P<0.01 for all). However, the nuclear expression of β-catenin was down-regulated (P<0.01). The NSC67657 treatment induced nonsignificant alterations of TCF-4 mRNA, total protein and nuclear protein in the HL-60 cells (P>0.05 for all). The target genes of Wnt signaling pathway, including c-myc, cyclinD1 and TCF-1 mRNA and proteins in the HL-60 cells were significantly down-regulated after NSC67657 treatment (P<0.05). The new steroidal drug NSC67657 induces monocytic differentiation of HL-60 cells, and down-regulates the expression of β-catenin and target genes of Wnt signaling pathway. These results indicate that Wnt signaling pathway may be directly or indirectly involved in the monocytic differentiation process of HL-60 cells.

Investigating Steroid Receptor Coactivator 3 (SRC3) as a Potential Therapeutic Target for Treating Advanced Prostate Cancer

DTIC Science & Technology

2013-04-01

to be a target of CHIP and knockdown of SRC-3 reduces Smad and Twist expression [81]. In human hepatocellular carcinoma , Hepatitis B virus X protein...stabilizes AIB1 protein and cooperates with it to promote human hepatocellular carcinoma cell invasiveness. Hepatology 2012. [Epub ahead of print] 83...amplification in hepatocellular carcinoma . A broad survey using high-throughput tissue microarray. Cancer 2002;95(11):2346-52 104. Xu Y, Chen Q, Li W, et al

Integrated analysis of zone-specific protein and metabolite profiles within nitrogen-fixing Medicago truncatula-Sinorhizobium medicae nodules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogden, Aaron J.; Gargouri, Mahmoud; Park, JeongJin

Symbiotic nitrogen fixation (SNF) between rhizobia and legumes requires metabolic coordination within specialized root organs called nodules. Nodules formed in the symbiosis between S. medicae and barrel medic (M. truncatula) are indeterminate, cylindrical, and contain spatially distinct developmental zones. Bacteria in the infection zone II (ZII), interzone II-III (IZ), and nitrogen fixation zone III (ZIII) represent different stages in the metabolic progression from free-living bacteria into nitrogen fixing bacteroids. To better understand the coordination of plant and bacterial metabolism within the nodule, we used liquid and gas chromatography coupled to tandem mass spectrometry (MS) to observe protein and metabolite profilesmore » representative of ZII, IZ, ZIII, whole-nodule, and primary root. Our MS-based approach confidently identified 361 S. medicae proteins and 888 M. truncatula proteins, as well as 160 metabolites from each tissue. The data are consistent with several organ- and zone-specific protein and metabolite localization patterns characterized previously. Here, we used our comprehensive dataset to demonstrate how multiple branches of primary metabolism are coordinated between symbionts and zones, including central carbon, fatty acid, and amino acid metabolism. For example, M. truncatula glycolysis enzymes accumulate from zone I to zone III within the nodule, while equivalent S. medicae enzymes decrease in abundance. We also show the localization of S. medicae's transition to dicarboxylic acid-dependent carbon metabolism within the IZ. The spatial abundance patterns of S. medicae fatty acid (FA) biosynthesis enzymes indicate an increased demand for FA production in the IZ and ZIII as compared to ZI. Our observations provide a resource for those seeking to understand coordinated physiological changes during the development of SNF.« less

Integrated analysis of zone-specific protein and metabolite profiles within nitrogen-fixing Medicago truncatula-Sinorhizobium medicae nodules

DOE PAGES

Ogden, Aaron J.; Gargouri, Mahmoud; Park, JeongJin; ...

2017-07-10

Symbiotic nitrogen fixation (SNF) between rhizobia and legumes requires metabolic coordination within specialized root organs called nodules. Nodules formed in the symbiosis between S. medicae and barrel medic (M. truncatula) are indeterminate, cylindrical, and contain spatially distinct developmental zones. Bacteria in the infection zone II (ZII), interzone II-III (IZ), and nitrogen fixation zone III (ZIII) represent different stages in the metabolic progression from free-living bacteria into nitrogen fixing bacteroids. To better understand the coordination of plant and bacterial metabolism within the nodule, we used liquid and gas chromatography coupled to tandem mass spectrometry (MS) to observe protein and metabolite profilesmore » representative of ZII, IZ, ZIII, whole-nodule, and primary root. Our MS-based approach confidently identified 361 S. medicae proteins and 888 M. truncatula proteins, as well as 160 metabolites from each tissue. The data are consistent with several organ- and zone-specific protein and metabolite localization patterns characterized previously. Here, we used our comprehensive dataset to demonstrate how multiple branches of primary metabolism are coordinated between symbionts and zones, including central carbon, fatty acid, and amino acid metabolism. For example, M. truncatula glycolysis enzymes accumulate from zone I to zone III within the nodule, while equivalent S. medicae enzymes decrease in abundance. We also show the localization of S. medicae's transition to dicarboxylic acid-dependent carbon metabolism within the IZ. The spatial abundance patterns of S. medicae fatty acid (FA) biosynthesis enzymes indicate an increased demand for FA production in the IZ and ZIII as compared to ZI. Our observations provide a resource for those seeking to understand coordinated physiological changes during the development of SNF.« less

Rac1 Recruitment to the Archipelago Structure of the Focal Adhesion through the Fluid Membrane as Revealed by Single-Molecule Analysis

PubMed Central

Shibata, Akihiro C E; Chen, Limin H; Nagai, Rie; Ishidate, Fumiyoshi; Chadda, Rahul; Miwa, Yoshihiro; Naruse, Keiji; Shirai, Yuki M; Fujiwara, Takahiro K; Kusumi, Akihiro

2013-01-01

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands. © 2013 Wiley Periodicals, Inc PMID:23341328

The Disease Protein Tulp1 Is Essential for Periactive Zone Endocytosis in Photoreceptor Ribbon Synapses

PubMed Central

Wahl, Silke; Magupalli, Venkat Giri; Dembla, Mayur; Katiyar, Rashmi; Schwarz, Karin; Köblitz, Louise; Alpadi, Kannan; Krause, Elmar; Rettig, Jens; Sung, Ching-Hwa; Goldberg, Andrew F. X.

2016-01-01

Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) in humans. In the retina, Tulp1 is mainly expressed in photoreceptors that use ribbon synapses to communicate with the inner retina. In the present study, we demonstrate that Tulp1 is highly enriched in the periactive zone of photoreceptor presynaptic terminals where Tulp1 colocalizes with major endocytic proteins close to the synaptic ribbon. Analyses of Tulp1 knock-out mice demonstrate that Tulp1 is essential to keep endocytic proteins enriched at the periactive zone and to maintain high levels of endocytic activity close to the synaptic ribbon. Moreover, we have discovered a novel interaction between Tulp1 and the synaptic ribbon protein RIBEYE, which is important to maintain synaptic ribbon integrity. The current findings suggest a new model for Tulp1-mediated localization of the endocytic machinery at the periactive zone of ribbon synapses and offer a new rationale and mechanism for vision loss associated with genetic defects in Tulp1. SIGNIFICANCE STATEMENT Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) and Leber congenital amaurosis (LCA15) in human patients. In this study, we discovered that the phosphoinositol-4,5-bisphosphate-binding protein Tulp1 is essential for the structural and functional organization of the periactive zone in photoreceptor synapses. Using Tulp1 knock-out mice, we found that Tulp1 is required to enrich major endocytic proteins at the periactive zone next to the synaptic ribbon. We demonstrate that Tulp1 is needed to promote endocytic vesicle retrieval at the periactive zone. Moreover, we discovered a novel interaction between Tulp1 and the synaptic ribbon protein RIBEYE. This newly discovered disease-sensitive interaction provides a molecular model for the control of endocytosis close to the synaptic ribbon. PMID:26911694

Clarinet (CLA-1), a novel active zone protein required for synaptic vesicle clustering and release

PubMed Central

Nelson, Jessica; Richmond, Janet E; Colón-Ramos, Daniel A; Shen, Kang

2017-01-01

Active zone proteins cluster synaptic vesicles at presynaptic terminals and coordinate their release. In forward genetic screens, we isolated a novel Caenorhabditis elegans active zone gene, clarinet (cla-1). cla-1 mutants exhibit defects in synaptic vesicle clustering, active zone structure and synapse number. As a result, they have reduced spontaneous vesicle release and increased synaptic depression. cla-1 mutants show defects in vesicle distribution near the presynaptic dense projection, with fewer undocked vesicles contacting the dense projection and more docked vesicles at the plasma membrane. cla-1 encodes three isoforms containing common C-terminal PDZ and C2 domains with homology to vertebrate active zone proteins Piccolo and RIM. The C-termini of all isoforms localize to the active zone. Specific loss of the ~9000 amino acid long isoform results in vesicle clustering defects and increased synaptic depression. Our data indicate that specific isoforms of clarinet serve distinct functions, regulating synapse development, vesicle clustering and release. PMID:29160205

Effects of 18beta-glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells.

PubMed

Huang, Shih-Horng; Wu, Jiahn-Chun; Hwang, Ra-Der; Yeo, Hui-Lin; Wang, Seu-Mei

2003-09-01

Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to alpha-, beta-, and gamma-catenin, which were seen as continuous, linear staining of cell-cell adherens junction. Treatment with 18beta-glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time- and dose-dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell-cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18beta-glycyrrhetinic acid stimulated a two- to three-fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18beta-glycyrrhetinic acid was partially mediated by extracellular signal-related kinase and calcium/calmodulin-dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells. Copyright 2003 Wiley-Liss, Inc.

Hsp90 can Accommodate the Simultaneous Binding of the FKBP52 and HOP Proteins

PubMed Central

Hildenbrand, Zacariah L.; Molugu, Sudheer K.; Herrera, Nadia; Ramirez, Citlally; Xiao, Chuan; Bernal, Ricardo A.

2011-01-01

The regulation of steroidogenic hormone receptor-mediated activity plays an important role in the development of hormone-dependent cancers. For example, during prostate carcinogenesis, the regulatory function played by the androgen receptor is often converted from a growth suppressor to an oncogene thus promoting prostate cancer cell survival and eventual metastasis. Within the cytoplasm, steroid hormone receptor activity is regulated by the Hsp90 chaperone in conjunction with a series of co-chaperone proteins. Collectively, Hsp90 and its binding associates form a large heteromeric complex that scaffold the fully mature receptor for binding with the respective hormone. To date our understanding of the interactions between Hsp90 with the various TPR domain-containing co-chaperone proteins is limited due to a lack of available structural information. Here we present the stable formation of Hsp902-FKBP521- HOP2 and Hsp902-FKBP521-p232-HOP2 complexes as detected by immunoprecipitation, time course dynamic light scattering and electron microscopy. The simultaneous binding of FKBP52 and HOP to the Hsp90 dimer provide direct evidence of a novel chaperone sub-complex that likely plays a transient role in the regulation of the fully mature steroid hormone receptor. PMID:21378414

Drug/protein interactions studied by time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Gustavsson, Thomas; Markovitsi, Dimitra; Vayá, Ignacio; Bonancía, Paula; Jiménez, M. C.; Miranda, Miguel A.

2014-09-01

We report here on a recent time-resolved fluorescence study [1] of the interaction between flurbiprofen (FBP), a chiral non-steroidal anti-inflammatory drug, and human serum albumin (HSA), the main transport protein in the human body. We compare the results obtained for the drug-protein complex with those of various covalently linked flurbiprofentryptophan dyads having well-defined geometries. In all cases stereoselective dynamic fluorescence quenching is observed, varying greatly from one system to another. In addition, the fluorescence anisotropy decays also display a clear stereoselectivity. For the drug-protein complexes, this can be interpreted in terms of the protein microenvironment playing a significant role in the conformational relaxation of FBP, which is more restricted in the case of the (R)- enantiomer.

Presynaptic active zones of mammalian neuromuscular junctions: Nanoarchitecture and selective impairments in aging.

PubMed

Badawi, Yomna; Nishimune, Hiroshi

2018-02-01

Neurotransmitter release occurs at active zones, which are specialized regions of the presynaptic membrane. A dense collection of proteins at the active zone provides a platform for molecular interactions that promote recruitment, docking, and priming of synaptic vesicles. At mammalian neuromuscular junctions (NMJs), muscle-derived laminin β2 interacts with presynaptic voltage-gated calcium channels to organize active zones. The molecular architecture of presynaptic active zones has been revealed using super-resolution microscopy techniques that combine nanoscale resolution and multiple molecular identification. Interestingly, the active zones of adult NMJs are not stable structures and thus become impaired during aging due to the selective degeneration of specific active zone proteins. This review will discuss recent progress in the understanding of active zone nanoarchitecture and the mechanisms underlying active zone organization in mammalian NMJs. Furthermore, we will summarize the age-related degeneration of active zones at NMJs, and the role of exercise in maintaining active zones. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Recent Advances in Drug Design and Drug Discovery for Androgen-Dependent Diseases

PubMed Central

Cabeza, Marisa; Sánchez-Márquez, Araceli; Garrido, Mariana; Silva, Aylín; Bratoeff, Eugene

2016-01-01

This article summarizes the importance of different targets such as 5α-reductase, 17β-HSD, CYP17A, androgen receptor and protein kinase A for the treatment of prostate cancer and benign prostatic hyperplasia. It is a well known fact that dihydrotestosterone (DHT) is associated with the development of androgen-dependent afflictions. At the present time, several research groups are attempting to develop new steroidal and non-steroidal molecules with the purpose of inhibiting the synthesis and biological response of DHT. This review also discusses the most recent studies reported in the literature that describe the therapeutic potential of novel compounds, as well as the new drugs, principally inhibitors of 5α-reductase. PMID:26861003

Is non‐steroidal anti‐inflammaory drug (NSAID) enteropathy clinically more important than NSAID gastropathy?

PubMed Central

Adebayo, D; Bjarnason, I

2006-01-01

The side effects of conventional non‐steroidal anti‐inflammatory drugs (NSAIDs) on the stomach is undoubtedly a serious public health problem contributing significantly to the morbidity and mortality of patients receiving these drugs. However, the damage of NSAIDs is not confined to the stomach. Indeed the short term and long term damage of NSAIDs on the small bowel (NSAID enteropathy) is more frequent than NSAID gastropathy. Furthermore, NSAID enteropathy is associated with complications (bleeding and protein loss). While many of these are mild, the serious events (significant bleeding, perforation, obstruction, and sudden death) are frequent as that reported for NSAID gastropathy. The diagnosis of NSAID enteropathy has been greatly aided by the introduction of wireless capsule enteroscopy. PMID:16517800

Is non-steroidal anti-inflammaory drug (NSAID) enteropathy clinically more important than NSAID gastropathy?

PubMed

Adebayo, D; Bjarnason, I

2006-03-01

The side effects of conventional non-steroidal anti-inflammatory drugs (NSAIDs) on the stomach is undoubtedly a serious public health problem contributing significantly to the morbidity and mortality of patients receiving these drugs. However, the damage of NSAIDs is not confined to the stomach. Indeed the short term and long term damage of NSAIDs on the small bowel (NSAID enteropathy) is more frequent than NSAID gastropathy. Furthermore, NSAID enteropathy is associated with complications (bleeding and protein loss). While many of these are mild, the serious events (significant bleeding, perforation, obstruction, and sudden death) are frequent as that reported for NSAID gastropathy. The diagnosis of NSAID enteropathy has been greatly aided by the introduction of wireless capsule enteroscopy.

Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xun, C.Q.; Ensor, C.M.; Tai, H.H.

1991-06-28

Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and (35S)methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.

Treatment of idiopathic FSGS with adrenocorticotropic hormone gel.

PubMed

Hogan, Jonathan; Bomback, Andrew S; Mehta, Kshama; Canetta, Pietro A; Rao, Maya K; Appel, Gerald B; Radhakrishnan, Jai; Lafayette, Richard A

2013-12-01

Adrenocorticotropic hormone (ACTH) has shown efficacy as primary and secondary therapy for nephrotic syndrome due to membranous nephropathy. The data on using ACTH to treat idiopathic FSGS are limited. This report describes our experience using ACTH for nephrotic syndrome due to idiopathic FSGS in the United States. Twenty-four patients with nephrotic syndrome from idiopathic FSGS were treated with ACTH gel at two academic medical centers between 2009 and 2012, either as part of investigator-initiated pilot studies (n=16) or by prescription for treatment-resistant FSGS (n=8). The primary outcome was remission of proteinuria. The median dose of ACTH was 80 units injected subcutaneously twice weekly. Treatment durations were not uniform. Twenty-two patients had received immunosuppression (mean, 2.2 medications) before ACTH therapy. Six patients had steroid-dependent and 15 had steroid-resistant FSGS. At the time of ACTH initiation, the median serum creatinine (interquartile range) was 2.0 (1.1-2.7) mg/dl, estimated GFR was 36 (28-78) ml/min per 1.73 m(2), and urine protein-to-creatinine ratio was 4595 (2200-8020) mg/g. At the end of ACTH therapy, 7 of 24 patients (29%) experienced remission (n=2 complete remissions, n=5 partial remissions). All remitters had steroid-resistant (n=5) or steroid-dependent (n=2) FSGS. Two responders relapsed during the follow-up period (mean ± SD, 70±31 weeks). Adverse events occurred in 21 of 24 patients, including one episode of new-onset diabetes that resolved after stopping ACTH and two episodes of AKI. Response to ACTH treatment among steroid-resistant or steroid-dependent patients with FSGS is low, but ACTH gel may be a viable treatment option for some patients with resistant nephrotic syndrome due to idiopathic FSGS. Further research is necessary to determine which patients will respond to therapy.

Treatment of Idiopathic FSGS with Adrenocorticotropic Hormone Gel

PubMed Central

Bomback, Andrew S.; Mehta, Kshama; Canetta, Pietro A.; Rao, Maya K.; Appel, Gerald B.; Radhakrishnan, Jai; Lafayette, Richard A.

2013-01-01

Summary Background and objectives Adrenocorticotropic hormone (ACTH) has shown efficacy as primary and secondary therapy for nephrotic syndrome due to membranous nephropathy. The data on using ACTH to treat idiopathic FSGS are limited. This report describes our experience using ACTH for nephrotic syndrome due to idiopathic FSGS in the United States. Design, setting, participants, & measurements Twenty-four patients with nephrotic syndrome from idiopathic FSGS were treated with ACTH gel at two academic medical centers between 2009 and 2012, either as part of investigator-initiated pilot studies (n=16) or by prescription for treatment-resistant FSGS (n=8). The primary outcome was remission of proteinuria. The median dose of ACTH was 80 units injected subcutaneously twice weekly. Treatment durations were not uniform. Results Twenty-two patients had received immunosuppression (mean, 2.2 medications) before ACTH therapy. Six patients had steroid-dependent and 15 had steroid-resistant FSGS. At the time of ACTH initiation, the median serum creatinine (interquartile range) was 2.0 (1.1–2.7) mg/dl, estimated GFR was 36 (28–78) ml/min per 1.73 m2, and urine protein-to-creatinine ratio was 4595 (2200–8020) mg/g. At the end of ACTH therapy, 7 of 24 patients (29%) experienced remission (n=2 complete remissions, n=5 partial remissions). All remitters had steroid-resistant (n=5) or steroid-dependent (n=2) FSGS. Two responders relapsed during the follow-up period (mean ± SD, 70±31 weeks). Adverse events occurred in 21 of 24 patients, including one episode of new-onset diabetes that resolved after stopping ACTH and two episodes of AKI. Conclusions Response to ACTH treatment among steroid-resistant or steroid-dependent patients with FSGS is low, but ACTH gel may be a viable treatment option for some patients with resistant nephrotic syndrome due to idiopathic FSGS. Further research is necessary to determine which patients will respond to therapy. PMID:24009220

Research on soybean protein wastewater treatment by the integrated two-phase anaerobic reactor

PubMed Central

Yu, Yaqin

2015-01-01

The start-up tests of treating soybean protein wastewater by the integrated two-phase anaerobic reactor were studied. The results showed that the soybean protein wastewater could be successfully processed around 30 days when running under the situation of dosing seed sludge with the influent of approximately 2000 mg/L and an HRT of 40 h. When the start-up was finished, the removal rate of COD by the reactor was about 80%. In the zone I, biogas mainly revealed carbon dioxide (CO2) and hydrogen (H2). Methane was the main component in the zone 2 which ranged from 53% to 59% with an average of 55%. The methane content in biogas increased from the zone I to II. It indicated that the methane-producing capacity of the anaerobic sludge increased. It was found that the uniquely designed two-phase integrated anaerobic reactor played a key role in treating soybean protein wastewater. The acidogenic fermentation bacteria dominated in the zone I, while methanogen became dominant in the zone II. It realized the relatively effective separation of hydrolysis acidification and methanogenesis process in the reactor, which was benefit to promote a more reasonable space distribution of the microbial communities in the reactor. There were some differences between the activities of the sludge in the two reaction zones of the integrated two-phase anaerobic reactor. The activity of protease was higher in the reaction zone I. And the coenzyme F420 in the reaction zone II was twice than that in the reaction zone I, which indicated that the activity of the methanogens was stronger in the reaction zone II. PMID:26288554

Spatially resolving the secretome within the mycelium of the cell factory Aspergillus niger.

PubMed

Krijgsheld, Pauline; Altelaar, A F Maarten; Post, Harm; Ringrose, Jeffrey H; Müller, Wally H; Heck, Albert J R; Wösten, Han A B

2012-05-04

Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature.

Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hudson, William H.; Pickard, Mark R.; de Vera, Ian Mitchelle S.

2014-12-23

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5more » lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.« less

FadA5 a thiolase from Mycobacterium tuberculosis - a unique steroid-binding pocket reveals the potential for drug development against tuberculosis

PubMed Central

Schaefer, Christin M.; Lu, Rui; Nesbitt, Natasha M.; Schiebel, Johannes; Sampson, Nicole S.; Kisker, Caroline

2014-01-01

Summary With the exception of HIV, tuberculosis (TB) is the leading cause of mortality among infectious diseases. The urgent need to develop new anti-tubercular drugs is apparent due to the increasing number of drug resistant Mycobacterium tuberculosis (Mtb) strains. Proteins involved in cholesterol import and metabolism have recently been discovered as potent targets against TB. FadA5, a thiolase from Mtb, is catalyzing the last step of the β-oxidation reaction of the cholesterol side-chain degradation under release of critical metabolites and was shown to be of importance during the chronic stage of TB infections. To gain structural and mechanistic insight on FadA5 we characterized the enzyme in different stages of the cleavage reaction and with a steroid bound to the binding pocket. Structural comparisons to human thiolases revealed that it should be possible to target FadA5 specifically and the steroid-bound structure provides a solid basis for the development of inhibitors against FadA5. PMID:25482540

Domestic dengue infection with hemophagocytic lymphohistiocytosis successfully treated by early steroid therapy.

PubMed

Yoshifuji, Kota; Oshina, Takahiro; Sonokawa, Saeko; Noguchi, Yuma; Suzuki, Sayaka; Tanaka, Keisuke; Kumagai, Takashi

2016-07-01

A 34-year-old man, working at a park in Tokyo, Japan, was repeatedly bitten by mosquitoes while cutting grass. He was hospitalized with sudden fever, fatigue, and weakness. He was eventually diagnosed with dengue virus infection, detected using reverse transcription polymerase chain reaction for the genome and by the presence of nonstructural protein 1 in his peripheral blood. Symptomatic treatments such as acetaminophen for the fever were not effective. Moreover, peripheral blood examination showed drastically decreased white blood cells and platelets, as well as marked elevations of ferritin and soluble interleukin 2 receptor. Furthermore, bone marrow examination revealed increased macrophages with hemophagocytosis. Dengue infection with hemophagocytic lymphohistiocytosis (HLH) was ultimately diagnosed. Half-dose steroid pulse therapy for three days dramatically reduced his temperature, thereby ameliorating physical symptoms and restoring normal peripheral blood data. He was discharged 12 days after admission. Dengue infection with HLH is rare and this is the first report, to our knowledge, of domestic dengue infection with HLH in Japan. Early steroid therapy may be effective in such cases.

Cardiotonic steroids-mediated Na+/K+-ATPase targeting could circumvent various chemoresistance pathways.

PubMed

Mijatovic, Tatjana; Kiss, Robert

2013-03-01

Many cancer patients fail to respond to chemotherapy because of the intrinsic resistance of their cancer to pro-apoptotic stimuli or the acquisition of the multidrug resistant phenotype during chronic treatment. Previous data from our groups and from others point to the sodium/potassium pump (the Na+/K+-ATPase, i.e., NaK) with its highly specific ligands (i.e., cardiotonic steroids) as a new target for combating cancers associated with dismal prognoses, including gliomas, melanomas, non-small cell lung cancers, renal cell carcinomas, and colon cancers. Cardiotonic steroid-mediated Na+/K+-ATPase targeting could circumvent various resistance pathways. The most probable pathways include the involvement of Na+/K+-ATPase β subunits in invasion features and Na+/K+-ATPase α subunits in chemosensitisation by specific cardiotonic steroid-mediated apoptosis and anoïkis-sensitisation; the regulation of the expression of multidrug resistant-related genes; post-translational regulation, including glycosylation and ubiquitinylation of multidrug resistant-related proteins; c-Myc downregulation; hypoxia-inducible factor downregulation; NF-κB downregulation and deactivation; the inhibition of the glycolytic pathway with a reduction of intra-cellular ATP levels and an induction of non-apoptotic cell death. The aims of this review are to examine the various molecular pathways by which the NaK targeting can be more deleterious to biologically aggressive cancer cells than to normal cells. Georg Thieme Verlag KG Stuttgart · New York.

Validation of murine and human placental explant cultures for use in sex steroid and phase II conjugation toxicology studies

PubMed Central

Sato, Brittany L.; Ward, Monika A.; Astern, Joshua M.; Kendal-Wright, Claire E.; Collier, Abby C.

2014-01-01

Human primary placental explant culture is well established for cytokine signaling and toxicity, but has not been validated for steroidogenic or metabolic toxicology. The technique has never been investigated in the mouse. We characterized human and mouse placental explants for up to 96hr in culture. Explant viability (Lactate dehydrogenase) and sex steroid levels were measured in media using spectrophotometry and ELISA, respectively. Expression and activities of the steroidogenic (3β-hydroxysteroid dehydrogenase, Cytochrome P45017A1, Cytochrome P45019), conjugation (UDP-glucuronosyltransferase, sulfotransferase (SULT)), and regeneration (β-glucuronidase, arylsulfatase C (ASC)) enzymes were determined biochemically in tissues with fluorimetric and spectrophotometric assays, and western blot. Explants were viable up to 96hr, but progesterone, estrone, and 17β-estradiol secretion decreased. Steroidogenic enzyme expression and activities were stable in mouse explants and similar to levels in freshly isolated tissues, but were lower in human explants than in fresh tissue (P<0.01). Human and mouse explants exhibited significantly less conjugation after 96hr, SULT was not detected in the mouse, and neither explants had active ASC, although proteins were expressed. Mouse explants may be useful for steroid biochemistry and endocrine disruption studies, but not metabolic conjugation. In contrast, human explants may be useful for studying conjugation for <48hr, but not for steroid/endocrine studies. PMID:25283089

Pharmacogenetics of steroid-responsive acute graft-versus-host disease.

PubMed

Arora, Mukta; Weisdorf, Daniel J; Shanley, Ryan M; Thyagarajan, Bharat

2017-05-01

Glucocorticoids are central to effective therapy of acute graft-versus-host disease (GVHD). However, only about half of the patients respond to steroids in initial therapy. Based on postulated mechanisms for anti-inflammatory effectiveness, we explored genetic variations in glucocorticoid receptor, co-chaperone proteins, membrane transporters, inflammatory mediators, and variants in the T-cell receptor complex in hematopoietic cell transplant recipients with acute GVHD requiring treatment with steroids and their donors toward response at day 28 after initiation of therapy. A total of 300 recipient and donor samples were analyzed. Twenty-three SNPs in 17 genes affecting glucocorticoid pathways were included in the analysis. In multiple regression analysis, donor SNP rs3192177 in the ZAP70 gene (O.R. 2.8, 95% CI: 1.3-6.0, P=.008) and donor SNP rs34471628 in the DUSPI gene (O.R. 0.3, 95% CI: 0.1-1.0, P=.048) were significantly associated with complete or partial response. However, after adjustment for multiple testing, these SNPs did not remain statistically significant. Our results, on this small, exploratory, hypothesis generating analysis suggest that common genetic variation in glucocorticoid pathways may help identify subjects with differential response to glucocorticoids. This needs further assessment in larger datasets and if validated could help identify subjects for alternative treatments and design targeted treatments to overcome steroid resistance. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Steroid instillation for idiopathic urethritis in children: an 8-year experience.

PubMed

Jayakumar, Sivasankar; Ninan, George Kaithayil; Pringle, Kirsty

2015-04-01

Idiopathic urethritis (IU) in children is of unknown etiology and treatment options are limited. We aim to report our experience with steroid instillation in IU in children. Retrospective data collection of all male children diagnosed with IU over a period of 8 years. Patients with balanitis xerotica obliterans (BXO) and positive urine culture at presentation were excluded from the study. Data were collected on patient demographics, laboratory and radiological investigations, cystoscopy findings, management, and outcomes. A total of 16 male children were diagnosed with IU. The mean age was 11.6 (7-16) years. Presenting symptoms included dysuria in 10; frank hematuria in 7; loin pain in 5; and scrotal pain in 2 patients. Serum C-reactive protein and full blood count was tested in 13 patients and was within normal limits in all of them. Endoscopy findings included posterior urethritis in 12, anterior urethritis in 2, and urethral stricture with inflammation in 2 patients. Ten patients required more than one episode of steroid instillation. Mean follow-up was 19.4 (1-74) months. Complete resolution of symptoms and signs occurred in 15 (93.6%) patients and improvement of symptoms and signs noted with ongoing treatment in 1 (6.4%) patient. IU in children can be successfully managed with steroid instillation. In our series, 93.6% of children had complete resolution of symptoms. Georg Thieme Verlag KG Stuttgart · New York.

Quantitative determination of testosterone levels with biolayer interferometry.

PubMed

Zhang, Hao; Li, Wei; Luo, Hong; Xiong, Guangming; Yu, Yuanhua

2017-10-01

Natural and synthetic steroid hormones are widely spread in the environment and are considered as pollutants due to their endocrine activities, even at low concentrations, which are harmful to human health. To detect steroid hormones in the environment, a novel biosensor system was developed based on the principle of biolayer interferometry. Detection is based on changes in the interference pattern of white light reflected from the surface of an optical fiber with bound biomolecules. Monitoring interactions between molecules does not require radioactive, enzymatic, or fluorescent labels. Here, 2 double-stranded DNA fragments of operator 1 (OP1) and OP2 containing 10-bp palindromic sequences in chromosomal Comamonas testosteroni DNA (ATCC11996) were surface-immobilized to streptavidin sensors. Interference changes were detected when repressor protein RepA bound the DNA sequences. DNA-protein interactions were characterized and kinetic parameters were obtained. The dissociation constants between the OP1 and OP2 DNA sequences and RepA were 9.865 × 10 -9  M and 2.750 × 10 -8  M, respectively. The reactions showed high specifically and affinity. Because binding of the 10-bp palindromic sequence and RepA was affected by RepA-testosterone binding, the steroid could be quantitatively determined rapidly using the biosensor system. The mechanism of the binding assay was as follows. RepA could bind both OP1 and testosterone. RepA binding to testosterone changed the protein conformation, which influenced the binding between RepA and OP1. The percentage of the signal detected negative correlation with the testosterone concentration. A standard curve was obtained, and the correlation coefficient value was approximately 0.97. We could quantitatively determine testosterone levels between 2.13 and 136.63 ng/ml. Each sample could be quantitatively detected in 17 min. These results suggested that the specific interaction between double-stranded OP1 DNA and the RepA protein could be used to rapidly and quantitatively determine environmental testosterone levels by the biolayer interferometry technique. Copyright © 2017 Elsevier B.V. All rights reserved.

Letrozole regulates actin cytoskeleton polymerization dynamics in a SRC-1 dependent manner in the hippocampus of mice.

PubMed

Zhao, Yangang; Yu, Yanlan; Zhang, Yuanyuan; He, Li; Qiu, Linli; Zhao, Jikai; Liu, Mengying; Zhang, Jiqiang

2017-03-01

In the hippocampus, local estrogens (E 2 ) derived from testosterone that is catalyzed by aromatase play important roles in the regulation of hippocampal neural plasticity, but the underlying mechanisms remain unclear. The actin cytoskeleton contributes greatly to hippocampal synaptic plasticity; however, whether it is regulated by local E 2 and the related mechanisms remain to be elucidated. In this study, we first examined the postnatal developmental profiles of hippocampal aromatase and specific proteins responsible for actin cytoskeleton dynamics. Then we used aromatase inhibitor letrozole (LET) to block local E 2 synthesis and examined the changes of these proteins and steroid receptor coactivator-1 (SRC-1), the predominant coactivator for steroid nuclear receptors. Finally, SRC-1 specific RNA interference was used to examine the effects of SRC-1 on the expression of these actin remodeling proteins. The results showed a V-type profile for aromatase and increased profiles for actin cytoskeleton proteins in both male and female hippocampus without obvious sex differences. LET treatment dramatically decreased the F-actin/G-actin ratio, the expression of Rictor, phospho-AKT (ser473), Profilin-1, phospho-Cofilin (Ser3), and SRC-1 in a dose-dependent manner. In vitro studies demonstrated that LET induced downregulation of these proteins could be reversed by E 2 , and E 2 induced increase of these proteins were significantly suppressed by SRC-1 shRNA interference. These results for the first time clearly demonstrated that local E 2 inhibition could induce aberrant actin polymerization; they also showed an important role of SRC-1 in the mediation of local E 2 action on hippocampal synaptic plasticity by regulation of actin cytoskeleton dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Computational Design of Ligand Binding Proteins with High Affinity and Selectivity

PubMed Central

Dou, Jiayi; Doyle, Lindsey; Nelson, Jorgen W.; Schena, Alberto; Jankowski, Wojciech; Kalodimos, Charalampos G.; Johnsson, Kai; Stoddard, Barry L.; Baker, David

2014-01-01

The ability to design proteins with high affinity and selectivity for any given small molecule would have numerous applications in biosensing, diagnostics, and therapeutics, and is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition phenomena. Attempts to design ligand binding proteins have met with little success, however, and the computational design of precise molecular recognition between proteins and small molecules remains an “unsolved problem”1. We describe a general method for the computational design of small molecule binding sites with pre-organized hydrogen bonding and hydrophobic interfaces and high overall shape complementary to the ligand, and use it to design protein binding sites for the steroid digoxigenin (DIG). Of 17 designs that were experimentally characterized, two bind DIG; the highest affinity design has the lowest predicted interaction energy and the most pre-organized binding site in the set. A comprehensive binding-fitness landscape of this design generated by library selection and deep sequencing was used to guide optimization of binding affinity to a picomolar level, and two X-ray co-crystal structures of optimized complexes show atomic level agreement with the design models. The designed binder has a high selectivity for DIG over the related steroids digitoxigenin, progesterone, and β-estradiol, which can be reprogrammed through the designed hydrogen-bonding interactions. Taken together, the binding fitness landscape, co-crystal structures, and thermodynamic binding parameters illustrate how increases in binding affinity can result from distal sequence changes that limit the protein ensemble to conformers making the most energetically favorable interactions with the ligand. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics, and diagnostics. PMID:24005320

Effect of omnivorous and vegan diets with different protein and carbohydrate content on growth and metabolism of growing rats.

PubMed

Giuberti, Gianluca; Morlacchini, Mauro; Crippa, Luca; Capraro, Jessica; Paganini, Beatrice; Gallo, Antonio; Rossi, Filippo

2018-08-01

The purpose of this study was to observe, in a rat animal model, the short and medium term effects of vegan (VEG) or omnivorous (OMNI) diets with different energy partition between nutrients (zone or classic). Six different diets were administered, for 72 days to 120 growing male Sprague-Dawley rats: (i) VEG zone diet; (ii) VEG classic diet; (iii) OMNI zone diet; (iv) OMNI classic diet; (v) OMNI zone diet with added fibre and (vi) OMNI classic diet with added fibre. Zone diets (high protein and low carbohydrates), resulted in better growth , feed efficiency, lower blood glucose and insulin responses. VEG diets have lowered cholesterol blood level. Histopathological analysis evidenced no damage to liver and kidney tissue by the intake of any of the diet types. Further longer animal and human duration studies should be performed to exclude detrimental effect of higher protein diet.

3D-QSAR Study of Steroidal and Azaheterocyclic Human Aromatase Inhibitors using Quantitative Profile of Protein-Ligand Interactions

EPA Science Inventory

Aromatase is a member of the cytochrome P450 superfamily responsible for a key step in the biosynthesis of estrogens. As estrogens are involved in the control of important reproduction-related processes, including sexual differentiation and maturation, aromatase is a potential ta...

Role of PY Motif Containing Protein, WBP-2 in ER, PR Signaling and Breast Tumorigenesis

DTIC Science & Technology

2009-09-01

WW-domains and PPXY motif have been implicated in many diseases, including muscular dystrophy , Liddle’s syndrome, Alzheimer’s, Huntington disease...ED, Becker M, Qiu Y, Johnson TA, Elbi C, Fletcher TM, John S, Hager GL 2004 Subnuclear trafficking and gene targeting by steroid re- ceptors. Ann NY

Dynamic Regulation of FoxA1 by Steroid Receptors | Center for Cancer Research

Cancer.gov

The estrogen receptor (ER) is a key regulator in breast cancer initiation and progression. A widely discussed model proposes that forkhead box protein A1 (FoxA1) acts as a pioneer factor in cancer by binding and penetrating closed chromatin to allow access by transcription factors (TFs), including ER.

The adolescent athlete and ergogenic aids.

PubMed

Dyment, P G

1987-01-01

Amphetamines and anabolic/androgenic steroids are the ergogenic aids with the most serious side effects that adolescents are likely to misuse, although adolescents are more frequently lured into taking expensive and worthless vitamin and protein supplements. Physicians performing preparticipation physical examinations should address the issue of ergogenic aids when they offer anticipatory guidance to adolescent athletes.

Obesity and Breast Cancer

DTIC Science & Technology

2005-07-01

serum INS, IGF-I and binding proteins, triglycerides, HDL - cholesterol , total and free steroids, sex hormone binding globulin, adiponectin, leptin, and...collection of information is estimated to average 1 hour per response , including the time for reviewing instructions, searching existing data sources...Bioinformatics, Biostatistics, Computer Science, Digital Mammography, Magnetic Resonance Imaging, Tissue Arrays, Gene Polymorphisms , Animal Models, Clinical

A study of the epitopes on steroid 21-hydroxylase recognized by autoantibodies in patients with or without Addison's disease

PubMed Central

Volpato, M; Prentice, L; Chen, S; Betterle, C; Rees Smith, B; Furmaniak, J

1998-01-01

Steroid 21-hydroxylase (21-OH) autoantibodies are found in patients with autoimmune Addison's disease (AAD), either isolated or associated with autoimmune polyglandular syndrome (APS) type I and II and in adrenal-cortex autoantibody (ACA)-positive patients without AAD. In order to assess any differences in the 21-OH autoantibodies in these different patient groups, we have studied their reactivity with different epitopes on 21-OH using full length and modified 35S-labelled 21-OH proteins produced in an in vitro transcription/translation system. There were no major differences in the pattern of autoantibody reactivity with the different modified 21-OH proteins in patients with isolated AAD or with APS types I and II, and in 21-OH autoantibody-positive patients with clinical AAD, subclinical AAD and those maintaining a normal adrenal function. Our studies also indicate that the main epitopes for 21-OH autoantibodies in patients with different forms of autoimmune adrenal disease are located in the C-terminal end and in a central region of 21-OH. PMID:9486414

How a RING finger protein and a steroid hormone control autophagy.

PubMed

Lindmo, Karine; Stenmark, Harald

2006-01-01

Previous work in our laboratory has indicated that the steroid hormone ecdysone triggers programmed autophagy in the fat body of Drosophila larvae by downregulating the class I phosphoinositide 3-kinase (PI3K) pathway. We recently found evidence that Deep orange (Dor), a Drosophila RING finger protein implicated in late-endosomal trafficking, controls ecdysone signaling as well as autolysosome fusion, thus exerting a dual regulation of autophagy. We found that dor mutants are defective in programmed autophagy. The mutant larvae showed impaired upregulation of ecdysone signaling during development, accompanied by a failure to downregulate the PI3K pathway. Downregulation of the PI3K pathway could be restored by feeding the dor mutants with ecdysone. Even though ecdysone signaling and autophagy were impaired in the dor mutants, we detected an accumulation of autophagosomes in dor mutant fat bodies. This could probably be attributed to the failure of autophagosomes to fuse with lysosomes. In this Addendum we review these findings and provide some speculations about how Dor may control both ecdysone signalling and autolysosomal fusion.

Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction

PubMed Central

Roche, John P.; Alsharif, Peter; Graf, Ethan R.

2015-01-01

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function. PMID:26317909

Crohn Disease: FDG PET/CT Before and After Initial Dose of Anti-Tumor Necrosis Factor Therapy to Predict Long-term Response.

PubMed

Epelboym, Yan; Shyn, Paul B; Chick, Jeffrey Forris Beecham; Hamilton, Matthew J; OʼConnor, Stacy D; Silverman, Stuart G; Kim, Chun K

2017-11-01

Clinical assessments of Crohn disease activity are limited in their capacity to assess treatment response to biologic therapy. The purpose of this study was to determine if changes in FDG activity between baseline PET and repeat PET performed prior to the second dose of induction anti-tumor necrosis factor (TNF) therapy correlate with clinical response. In this prospective, institutional review board-approved, Health Insurance Portability and Accountability Act-compliant pilot study of 8 adult patients with active Crohn disease, FDG activity before and 2 weeks after initiation of anti-TNF therapy was assessed using low-dose PET/CT. FDG activity was measured in the most inflamed bowel loop using an SUVratio (SUVmax/liver SUVmean). Changes in SUVratio were compared with a blinded gastroenterologist assessment of clinical response and steroid-free remission, as well as C-reactive protein (CRP), during a 12-month follow-up period. Of 8 patients, 7 showed FDG activity decline at 2 weeks, 5 of whom achieved a clinical response and steroid-free remission at 8, 26, and 52 weeks. The remaining 2 patients with FDG activity decline did not achieve a clinical response or steroid-free remission at these time points, but there were reductions in CRP. The 1 patient without FDG activity decline was a clinical nonresponder, had no reduction in CRP, and did not achieve steroid-free remission. A change in FDG activity at FDG PET/CT performed prior to the second induction dose of anti-TNF therapy has the potential to predict clinical response and steroid-free remission in patients with Crohn disease.

Study of body composition, lung function, and quality of life following use of anabolic steroids in patients with chronic obstructive pulmonary disease.

PubMed

Daga, Mradul Kumar; Khan, Naushad Ahmad; Malhotra, Varun; Kumar, Suman; Mawari, Govind; Hira, Harmanjit Singh

2014-04-01

Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow limitation and is associated with weight loss and decreased muscle strength and exercise capacity. A double-blinded randomized controlled trial of 32 male COPD patients (age, 54.94 ± 11.27 years) was carried out to assess effects of anabolic steroid in terms of a daily high-protein, high-calorie diet alone or one combined with anabolic steroids on body composition, lung function, and health-related quality of life (HRQL). Outcomes were assessed by anthropometric and spirometric measurements, peak expiratory flow rate, partial pressure of oxygen in arterial blood, 6-minute walk test (6MWT), hand grip test, and HRQL index scores. Measurements were made at baseline and end of treatment (6 weeks). All patients showed significant difference (P < .001) in pulmonary function parameters and anthropometric measurements after 6 weeks of intervention (within-group changes); however, no significant improvement occurred in the pulmonary function parameters between the groups. The difference in exercise capacity (6MWT) and HRQL scores in the treatment group were statistically significant (P < .001) compared with control group after 6 weeks of intervention. In the treatment group, the average 6MWT distance increased from 213.5 m to 268.5 m at 6-week follow-up, and HRQL scores increased from 101.25 to 118.45. Also, HRQL and 6MWT parameters were positively correlated in response to steroid supplementation at the end of the study. Weekly administration of anabolic steroids during 6 weeks increased exercise capacity and quality of life in patients with COPD.

Newborn lamb coronary artery reactivity is programmed by early gestation dexamethasone before the onset of systemic hypertension

PubMed Central

Roghair, Robert D.; Segar, Jeffrey L.; Sharma, Ram V.; Zimmerman, Matthew C.; Jagadeesha, D. K.; Segar, Emily M.; Scholz, Thomas D.; Lamb, Fred S.

2009-01-01

Exposure of the early gestation ovine fetus to exogenous glucocorticoids induces organ-specific alterations in postnatal cardiovascular physiology. To determine whether early gestation corticosteroid exposure alters coronary reactivity before the development of systemic hypertension, dexamethasone (0.28 mg·kg−1 · day−1) was administered to pregnant ewes by intravenous infusion over 48 h beginning at 27 days gestation (term, 145 days). Vascular responsiveness was assessed in endothelium-intact coronary arteries isolated from 1-wk-old steroid-exposed and age-matched control lambs (N = 6). Calcium imaging was performed in fura 2-loaded primary cultures of vascular smooth muscle cells (VSMC) from the harvested coronary arteries. Early gestation steroid exposure did not significantly alter mean arterial blood pressure or coronary reactivity to KCl, thromboxane A2 mimetic U-46619, or ANG II. Steroid exposure significantly increased coronary artery vasoconstriction to acetylcholine and endothelin-1. Vasodilatation to adenosine, but not nitroprusside or forskolin, was significantly attenuated following early gestation steroid exposure. Endothelin-1 or U-46619 stimulation resulted in a comparable increase in intracellular calcium concentration ([Ca2+]i) in coronary VSMC isolated from either dexamethasone-treated or control animals. However, the ANG II- or KCl-mediated increase in [Ca2+]i in control VSMC was significantly attenuated in VSMC harvested from dexamethasone-treated lambs. Coronary expression of muscle voltage-gated l-type calcium channel α-1 subunit protein was not significantly altered by steroid exposure, whereas endothelial nitric oxide synthase expression was attenuated. These findings demonstrate that early gestation glucocorticoid exposure elicits primary alterations in coronary responsiveness before the development of systemic hypertension. Glucocorticoid-induced alterations in coronary physiology may provide a mechanistic link between an adverse intrauterine environment and later cardiovascular disease. PMID:15961529

Novel CYP17 inhibitors: synthesis, biological evaluation, structure-activity relationships and modelling of methoxy- and hydroxy-substituted methyleneimidazolyl biphenyls.

PubMed

Hille, Ulrike E; Hu, Qingzhong; Vock, Carsten; Negri, Matthias; Bartels, Marc; Müller-Vieira, Ursula; Lauterbach, Thomas; Hartmann, Rolf W

2009-07-01

Recently, the steroidal CYP17 inhibitor Abiraterone entered phase II clinical trial for the treatment of androgen-dependent prostate cancer. As 17alpha-hydroxylase-17,20-lyase (CYP17) catalyzes the last step in androgen biosynthesis, inhibition of this target should affect not only testicular but also adrenal androgen formation. Therefore CYP17 inhibitors should be advantageous over existing therapies, for example with GnRH analogues. However, steroidal drugs are known for side effects which are due to affinities for steroid receptors. Therefore we decided to synthesize non-steroidal compounds mimicking the natural CYP17 substrates pregnenolone and progesterone. The synthesis and biological evaluation of a series of 15 novel and highly active non-steroidal CYP17 inhibitors are reported. The compounds were prepared via Suzuki-cross-coupling, Grignard reaction and CDI-assisted S(N)t-reaction with imidazole and their inhibitory activity was examined with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were further tested for their selectivity against the hepatic enzyme CYP3A4 and the glucocorticoid-forming enzyme CYP11B1. All compounds turned out to be potent CYP17 inhibitors. The most active compounds 7 and 8 were much more active than Ketoconazole showing activity comparable to Abiraterone (IC(50) values of 90 and 52nM vs. 72nM). Most compounds also showed higher selectivities than Ketoconazole, but turned out to be less selective than Abiraterone. Docking studies using our CYP17 protein model were performed with selected compounds to study the interactions between the inhibitors and the amino acid residues of the active site.

Studies on the antimicrobial effects of Spondias mombin and Baphia nittida on dental caries organism.

PubMed

Amadi, E S; Oyeka, A; Onyeagba, R A; Okoli, I; Ugbogu, O C

2007-02-01

The antimicrobial effect of cold water, hot water and ethanolic extracts of Spondias mombin and Baphia nittida on cariogenic streptococci isolated from dental caries patients attending the Ebonyi State University Teaching hospital dental clinic Abakaliki was investigated using the agar well diffusion technique. The cold water and ethanolic extracts of Baphia nittida showed inhibition zone diameter (IZD) of 10 and 12 mm respectively at 400 mg mL(-1), while the hot water showed no inhibitory effect. All extracts of Spondias mombin did not inhibit the test organism. The cold water and ethanolic extracts of Baphia nittida showed Minimum Inhibitory Concentration (MIC) of 100 and 50 mg mL(-1) respectively. The combination of the cold water extracts of the two herbs showed enhanced activity of 13 mm. Phytochemical analysis of Baphia nittida revealed the presence of flavonoids, glycosides, proteins saponins, tannins, carbohydrate and steroidal aglycone. Acute toxicity testing of Baphia nittida at a range of 250-5000 mg kg(-1) bw using mice showed no clinical signs of acute toxicity. No chemical toxicity was observed amongst rats given Baphia nittida extracts 500 and 1000 mg kg(-1) bw after 30 days. Baphia nittida may be a potential source of an antimicrobial agent for the treatment and management of dental caries.

Another cat and mouse game: Deciphering the evolution of the SCGB superfamily and exploring the molecular similarity of major cat allergen Fel d 1 and mouse ABP using computational approaches

PubMed Central

Pageat, Patrick; Bienboire-Frosini, Cécile

2018-01-01

The mammalian secretoglobin (SCGB) superfamily contains functionally diverse members, among which the major cat allergen Fel d 1 and mouse salivary androgen-binding protein (ABP) display similar subunits. We searched for molecular similarities between Fel d 1 and ABP to examine the possibility that they play similar roles. We aimed to i) cluster the evolutionary relationships of the SCGB superfamily; ii) identify divergence patterns, structural overlap, and protein-protein docking between Fel d 1 and ABP dimers; and iii) explore the residual interaction between ABP dimers and steroid binding in chemical communication using computational approaches. We also report that the evolutionary tree of the SCGB superfamily comprises seven unique palm-like clusters, showing the evolutionary pattern and divergence time tree of Fel d 1 with 28 ABP paralogs. Three ABP subunits (A27, BG27, and BG26) share phylogenetic relationships with Fel d 1 chains. The Fel d 1 and ABP subunits show similarities in terms of sequence conservation, identical motifs and binding site clefts. Topologically equivalent positions were visualized through superimposition of ABP A27:BG27 (AB) and ABP A27:BG26 (AG) dimers on a heterodimeric Fel d 1 model. In docking, Fel d 1-ABP dimers exhibit the maximum surface binding ability of AG compared with that of AB dimers and the several polar interactions between ABP dimers with steroids. Hence, cat Fel d 1 is an ABP-like molecule in which monomeric chains 1 and 2 are the equivalent of the ABPA and ABPBG monomers, respectively. These findings suggest that the biological and molecular function of Fel d 1 is similar to that of ABP in chemical communication, possibly via pheromone and/or steroid binding. PMID:29771985

Another cat and mouse game: Deciphering the evolution of the SCGB superfamily and exploring the molecular similarity of major cat allergen Fel d 1 and mouse ABP using computational approaches.

PubMed

Durairaj, Rajesh; Pageat, Patrick; Bienboire-Frosini, Cécile

2018-01-01

The mammalian secretoglobin (SCGB) superfamily contains functionally diverse members, among which the major cat allergen Fel d 1 and mouse salivary androgen-binding protein (ABP) display similar subunits. We searched for molecular similarities between Fel d 1 and ABP to examine the possibility that they play similar roles. We aimed to i) cluster the evolutionary relationships of the SCGB superfamily; ii) identify divergence patterns, structural overlap, and protein-protein docking between Fel d 1 and ABP dimers; and iii) explore the residual interaction between ABP dimers and steroid binding in chemical communication using computational approaches. We also report that the evolutionary tree of the SCGB superfamily comprises seven unique palm-like clusters, showing the evolutionary pattern and divergence time tree of Fel d 1 with 28 ABP paralogs. Three ABP subunits (A27, BG27, and BG26) share phylogenetic relationships with Fel d 1 chains. The Fel d 1 and ABP subunits show similarities in terms of sequence conservation, identical motifs and binding site clefts. Topologically equivalent positions were visualized through superimposition of ABP A27:BG27 (AB) and ABP A27:BG26 (AG) dimers on a heterodimeric Fel d 1 model. In docking, Fel d 1-ABP dimers exhibit the maximum surface binding ability of AG compared with that of AB dimers and the several polar interactions between ABP dimers with steroids. Hence, cat Fel d 1 is an ABP-like molecule in which monomeric chains 1 and 2 are the equivalent of the ABPA and ABPBG monomers, respectively. These findings suggest that the biological and molecular function of Fel d 1 is similar to that of ABP in chemical communication, possibly via pheromone and/or steroid binding.

A protein block based fold recognition method for the annotation of twilight zone sequences.

PubMed

Suresh, V; Ganesan, K; Parthasarathy, S

2013-03-01

The description of protein backbone was recently improved with a group of structural fragments called Structural Alphabets instead of the regular three states (Helix, Sheet and Coil) secondary structure description. Protein Blocks is one of the Structural Alphabets used to describe each and every region of protein backbone including the coil. According to de Brevern (2000) the Protein Blocks has 16 structural fragments and each one has 5 residues in length. Protein Blocks fragments are highly informative among the available Structural Alphabets and it has been used for many applications. Here, we present a protein fold recognition method based on Protein Blocks for the annotation of twilight zone sequences. In our method, we align the predicted Protein Blocks of a query amino acid sequence with a library of assigned Protein Blocks of 953 known folds using the local pair-wise alignment. The alignment results with z-value ≥ 2.5 and P-value ≤ 0.08 are predicted as possible folds. Our method is able to recognize the possible folds for nearly 35.5% of the twilight zone sequences with their predicted Protein Block sequence obtained by pb_prediction, which is available at Protein Block Export server.

Similarities between several major extinctions and preservation of life - biomolecules to geomolecules: an interdisciplinary approach

NASA Astrophysics Data System (ADS)

Grice, Kliti; Melendez, Ines; Tulipani, Svenja

2015-04-01

WA Organic and Isotope Geochemistry Centre, The Institute for Geoscience Research, Department of Chemistry, Curtin University, GPO Box U1987 Perth, WA 6845, Australia Photic zone euxinia in ancient seas has proven signficant for elucidating biogeochemical changes that occurred during three of the five Phanerozoic mass extinctions, viz. the Permian/Triassic [1], Triassic/Jurassic [2] and Late Givetian (Devonian) [3] events, including the conditions associated with exceptional fossil preservation [4,5]. The series of events preceding, during and post the Triassic/Jurassic event, is remarkably similar to that reported for the Permian/Triassic extinction, the largest of the Phanerozoic Era. For the Late Givetian event, the first forests evolved and reef-building communities and associated fauna in tropical, marine settings were largely affected [6]. Sedimentary rocks on the margins of the Devonian reef slope in the Canning Basin, WA, contain novel biomarker, isotopic and palynological evidence for the existence of a persistently stratified water-column (comprising a freshwater lens overlying a more saline hypolimnion), with prevailing anoxia and PZE [7]. Also from the Canning Basin, the exceptional preservation of a suite of biomarkers in a Devonian invertebrate fossil within a carbonate concretion supports rapid encasement of the crustacean (identified by % of C27 steroids) enhanced by sulfate reducing bacteria under PZE conditions. PZE plays a critical role in fossil (including soft tissue) and biomarker preservation. In the same sample, the oldest occurrence of intact sterols shows that they have been preserved for ca. 380 Ma [5]. The exceptional preservation of this biomass is attributed to microbially induced carbonate encapsulation, preventing full decomposition and transformation, thus extending the record of sterol occurrences in the geosphere by 250 Ma. A suite of ca. 50 diagenetic transformation products of sterols is also reported, showing the unique coexistence of biomolecules and geomolecules in the same sample, previously assumed unfeasible. The coexistence of steroids in a diagenetic continuum, ranging from stenols to triaromatic steroids, is attributed to microbially mediated eogenetic processes. Under exceptional conditions concretions preserve biomolecules at extraordinary levels, providing a new opportunity to study the distributions of biomolecules in deep time and thereby improving our understanding of the evolution of life where fossils are rarely preserved. References [1] Grice K., Cao C., Love G.D., Bottcher M.E., Twitchett R,, Grosjean E., Summons R.E., Turgeon S., Dunning W.J. & Jin Y., 2005. Photic zone euxinia during the Permian-Triassic superanoxic event. Science. 307, 706-709. [2] Jaraula C.M.B, Grice K., Twitchett R.,J., Böttcher M.E., Le Metayer P., Dastidar A.G. & Opazo L.F., 2013. Elevated pCO2 leading to Late triassic extinction, persistent photic zone euxinia, and rising sea levels. Geology 41, 955-958. [3] Tulipani S., Grice K., Greenwood P.F, Haines P., Sauer P., Schimmelmann A., Summons R.E., Foster C.B., Böttcher M.E., Playton, T. & Schwark L., 2014a. Changes in palaeoenvironmental conditions in Late Devonian Reef systems from the Canning Basin, WA: A biomarker and stable isotope approach. Gondwana Research, in press. [4] Melendez I., Grice K., Trinajstic K., Ladjavardi M., Thompson K. & Greenwood P.F., 2013a. Biomarkers reveal the role of photic zone euxinia in exceptional fossil preservation: An organic geochemical perspective. Geology 41, 123-126. [5] Melendez I., Grice K. & Schwark L., 2013b. Exceptional preservation of palaeozoic steroids in a diagenetic continuum. Nature Scientific Reports. 3. [6] Grice K., Lu H., Atahan P., Hallmann, C., Asif M., Greenwood P.F., Tulipani S., Maslen E., Williford W.H. & Dodson J. 2009. New insights into the origin of perlyene in geological samples Geochimica et Cosmochimica Acta, 73, 6531-6543. [7] Tulipani S., Grice K., Greenwood P.F., Schwark L., Summons R.E., Böttcher M.E. & Foster C.B., 2014b. Molecular proxies as indicators of freshwater incursion-driven salinity stratification. Geochemistry, Geophysics, Geosystems, accepted.

High-speed superresolution imaging of the proteins in fission yeast clathrin-mediated endocytic actin patches

PubMed Central

Arasada, Rajesh; Sayyad, Wasim A.; Berro, Julien; Pollard, Thomas D.

2018-01-01

To internalize nutrients and cell surface receptors via clathrin-mediated endocytosis, cells assemble at least 50 proteins, including clathrin, clathrin-interacting proteins, actin filaments, and actin binding proteins, in a highly ordered and regulated manner. The molecular mechanism by which actin filament polymerization deforms the cell membrane is unknown, largely due to lack of knowledge about the organization of the regulatory proteins and actin filaments. We used high-speed superresolution localization microscopy of live fission yeast cells to improve the spatial resolution to ∼35 nm with 1-s temporal resolution. The nucleation promoting factors Wsp1p (WASp) and Myo1p (myosin-I) define two independent pathways that recruit Arp2/3 complex, which assembles two zones of actin filaments. Myo1p concentrates at the site of endocytosis and initiates a zone of actin filaments assembled by Arp2/3 complex. Wsp1p appears simultaneously at this site but subsequently moves away from the cell surface as it stimulates Arp2/3 complex to assemble a second zone of actin filaments. Cells lacking either nucleation-promoting factor assemble only one, stationary, zone of actin filaments. These observations support our two-zone hypothesis to explain endocytic tubule elongation and vesicle scission in fission yeast. PMID:29212877

Impaired osteogenic differentiation associated with connexin43/microRNA-206 in steroid-induced avascular necrosis of the femoral head.

PubMed

Liu, Gang; Luo, Gaobin; Bo, Zhandong; Liang, Xiaonan; Huang, Jie; Li, Donghui

2016-08-01

Connexin(Cx)43 and microRNA(miR)-206 play an important role in osteogenesis. However, their role in steroid-induced femoral head osteonecrosis (SANFH) is still ambiguous. The present study aimed to establish a rabbit model and investigate osteogenesis in steroid-induced femoral head osteonecrosis occurring via Cx43/miR-206 and the changes of Wnt/β-catenin signal pathway-related proteins. A total of 72 adult New Zealand white rabbits were divided randomly into a model group (Group A) and a control group (Group B) of 36 rabbits each. Group A was injected intravenously with lipopolysaccharide (10μg/kg body weight, once per day). After 48h, three injections of methylprednisolone (MPS; 20mg/kg body weight) were administered intramuscularly at 24-hour intervals. Group B were fed and housed under identical conditions but received saline injections. All animals were sacrificed at two, four, and eight weeks from the first MPS injection. Typical early osteonecrosis symptoms were observed in Group A. The expression of miR-206 in Group A was significantly higher than that of Group B. The mRNA and protein levels of Cx43, β-catenin, runt-related transcription factor 2, and alkaline phosphatase gradually decreased while Dickkopf-1 (Dkk-1) gradually increased in Group A compared with Group B. These findings indicated that Cx43/miR-206 is involved in the pathogenesis of early stage SANFH and may be associate with Wnt/β-catenin signal pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism

PubMed Central

Lakisic, Goran; Wendling, Olivia; Libertini, Emanuele; Radford, Elizabeth J.; Le Guillou, Morwenna; Champy, Marie-France; Wattenhofer-Donzé, Marie; Soubigou, Guillaume; Ait-Si-Ali, Slimane; Feunteun, Jean; Sorg, Tania; Coppée, Jean-Yves; Ferguson-Smith, Anne C.; Cossart, Pascale; Bierne, Hélène

2016-01-01

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases. PMID:26938916

Cardiotonic steroid ouabain stimulates expression of blood-testis barrier proteins claudin-1 and -11 and formation of tight junctions in Sertoli cells.

PubMed

Dietze, Raimund; Shihan, Mazen; Stammler, Angelika; Konrad, Lutz; Scheiner-Bobis, Georgios

2015-04-15

The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the α4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Identification of 9α-Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

PubMed Central

Hayashi, Toshiaki; Koshino, Hiroyuki; Malon, Michal; Hirota, Hiroshi; Kudo, Toshiaki

2014-01-01

Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30. PMID:25092028

[The mammalian oviduct revisited].

PubMed

Halter, S; Reynaud, K; Tahir, Z; Thoumire, S; Chastant-Maillard, S; Saint-Dizier, M

2011-11-01

The oviducts, or uterine tubes, support the transport and final maturation of gametes, and harbour fertilization and early embryo development. The oviduct environment is finely regulated by ovarian steroids as well as by gametes and embryos that interact with it. Previously regarded as a simple transit zone, the oviduct is now regarded as a complex organ with multiple functions in these various processes. The tubal fluid, now better characterized, is to be regarded as the first interface between the mother and the embryo. It may play a major role in the quality of the conceptus. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries.

PubMed

Liu, Shaorong; Gao, Lin; Pu, Qiaosheng; Lu, Joann J; Wang, Xingjia

2006-02-01

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.

Experimental design based 3-D QSAR analysis of steroid-protein interactions: Application to human CBG complexes

NASA Astrophysics Data System (ADS)

Norinder, Ulf

1990-12-01

An experimental design based 3-D QSAR analysis using a combination of principal component and PLS analysis is presented and applied to human corticosteroid-binding globulin complexes. The predictive capability of the created model is good. The technique can also be used as guidance when selecting new compounds to be investigated.

Anticipatory UPR Activation: A Protective Pathway and Target in Cancer

PubMed Central

Shapiro, David J.; Livezey, Mara; Yu, Liqun; Zheng, Xiaobin; Andruska, Neal

2016-01-01

The endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), plays a key role in regulating intracellular protein homeostasis. The extensively studied reactive mode of UPR activation is characterized by unfolded protein, or other EnR stress, triggering UPR activation. Here we focus on the emerging anticipatory mode of UPR activation in which mitogenic steroid and peptide hormones and other effectors pre-activate the UPR and anticipate a future need for increased protein folding capacity. Mild UPR activation in breast cancer can be protective and contributes to antiestrogen resistance. Hyperactivation of the anticipatory UPR pathway in cancer cells with a small molecule converts it from cytoprotective to cytotoxic, highlighting its potential as a therapeutic target in estrogen receptor positive breast cancer. PMID:27354311

Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport.

PubMed

Barton, Chris; Beck, Paul; Kay, Richard; Teale, Phil; Roberts, Jane

2009-06-01

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race-horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis-driven discovery of doping biomarker candidates.

The twilight zone between protein order and disorder.

PubMed

Szilágyi, A; Györffy, D; Závodszky, P

2008-08-01

The amino acid composition of intrinsically disordered proteins and protein segments characteristically differs from that of ordered proteins. This observation forms the basis of several disorder prediction methods. These, however, usually perform worse for smaller proteins (or segments) than for larger ones. We show that the regions of amino acid composition space corresponding to ordered and disordered proteins overlap with each other, and the extent of the overlap (the "twilight zone") is larger for short than for long chains. To explain this finding, we used two-dimensional lattice model proteins containing hydrophobic, polar, and charged monomers and revealed the relation among chain length, amino acid composition, and disorder. Because the number of chain configurations exponentially grows with chain length, a larger fraction of longer chains can reach a low-energy, ordered state than do shorter chains. The amount of information carried by the amino acid composition about whether a protein or segment is (dis)ordered grows with increasing chain length. Smaller proteins rely more on specific interactions for stability, which limits the possible accuracy of disorder prediction methods. For proteins in the "twilight zone", size can determine order, as illustrated by the example of two-state homodimers.

Systemic SIRT1 insufficiency results in disruption of energy homeostasis and steroid hormone metabolism upon high-fat-diet feeding

PubMed Central

Purushotham, Aparna; Xu, Qing; Li, Xiaoling

2012-01-01

SIRT1 is a highly-conserved NAD+-dependent protein deacetylase that plays essential roles in the regulation of energy metabolism, genomic stability, and stress response. Although the functions of SIRT1 in many organs have been extensively studied in tissue-specific knockout mouse models, the systemic role of SIRT1 is still largely unknown as a result of severe developmental defects that result from whole-body knockout in mice. Here, we investigated the systemic functions of SIRT1 in metabolic homeostasis by utilizing a whole-body SIRT1 heterozygous mouse model. These mice are phenotypically normal under standard feeding conditions. However, when chronically challenged with a 40% fat diet, they become obese and insulin resistant, display increased serum cytokine levels, and develop hepatomegaly. Hepatic metabolomic analyses revealed that SIRT1 heterozygous mice have elevated gluconeogenesis and oxidative stress. Surprisingly, they are depleted of glycerolipid metabolites and free fatty acids, yet accumulate lysolipids. Moreover, high-fat feeding induces elevation of serum testosterone levels and enlargement of seminal vesicles in SIRT1 heterozygous males. Microarray analysis of liver mRNA indicates that they have altered expression of genes involved in steroid metabolism and glycerolipid metabolism. Taken together, our findings indicate that SIRT1 plays a vital role in the regulation of systemic energy and steroid hormone homeostasis.—Purushotham, A., Xu, Q., Li, X. Systemic SIRT1 insufficiency results in disruption of energy homeostasis and steroid hormone metabolism upon high-fat-diet feeding. PMID:22006157

Steroid receptor coactivator-3 regulates glucose metabolism in bladder cancer cells through coactivation of hypoxia inducible factor 1α.

PubMed

Zhao, Wei; Chang, Cunjie; Cui, Yangyan; Zhao, Xiaozhi; Yang, Jun; Shen, Lan; Zhou, Ji; Hou, Zhibo; Zhang, Zhen; Ye, Changxiao; Hasenmayer, Donald; Perkins, Robert; Huang, Xiaojing; Yao, Xin; Yu, Like; Huang, Ruimin; Zhang, Dianzheng; Guo, Hongqian; Yan, Jun

2014-04-18

Cancer cell proliferation is a metabolically demanding process, requiring high glycolysis, which is known as "Warburg effect," to support anabolic growth. Steroid receptor coactivator-3 (SRC-3), a steroid receptor coactivator, is overexpressed and/or amplified in multiple cancer types, including non-steroid targeted cancers, such as urinary bladder cancer (UBC). However, whether SRC-3 regulates the metabolic reprogramming for cancer cell growth is unknown. Here, we reported that overexpression of SRC-3 accelerated UBC cell growth, accompanied by the increased expression of genes involved in glycolysis. Knockdown of SRC-3 reduced the UBC cell glycolytic rate under hypoxia, decreased tumor growth in nude mice, with reduction of proliferating cell nuclear antigen and lactate dehydrogenase expression levels. We further revealed that SRC-3 could interact with hypoxia inducible factor 1α (HIF1α), which is a key transcription factor required for glycolysis, and coactivate its transcriptional activity. SRC-3 was recruited to the promoters of HIF1α-target genes, such as glut1 and pgk1. The positive correlation of expression levels between SRC-3 and Glut1 proteins was demonstrated in human UBC patient samples. Inhibition of glycolysis through targeting HK2 or LDHA decelerated SRC-3 overexpression-induced cell growth. In summary, overexpression of SRC-3 promoted glycolysis in bladder cancer cells through HIF1α to facilitate tumorigenesis, which may be an intriguing drug target for bladder cancer therapy.

Effects of mycoplasmal LAMPs on receptor responses to steroid hormones in mammalian cells.

PubMed

Iyama, K; Zhang, S; Lo, S C

2001-09-01

Many individuals are chronically infected or parasitically colonized with mycoplasmas in their respiratory or urogenital tracts without apparent clinical significance. However, prolonged close interaction between prokaryotic agents and eukaryotic host cells may gradually and significantly alter normal biological or physiological properties of infected hosts. Steroid hormones are associated with rates of cancer formation in human. The purpose of this study is to establish a sensitive reporting system to examine whether mycoplasmal infections affect biological responses to steroid hormones in mammalian cells. We established pMTV-CAT stably transfected cell lines to test the effect of mycoplasmal lipid-associated membrane proteins (LAMPs). Results showed that LAMPs (1 microg/ml) from seven different species of human mycoplasmas-M. penetrans, M. fermentans, M. genitalium, M. salivarium, M. pneumoniae, M. orale, and M. hominis-had an inhibitory effect on androgen receptor (AR) response to 5alpha-dihydrotestosterone (DHT) in the E82 transfectants. The inhibitory effect of mycoplasmal LAMPs appeared to be dose dependent. LAMPs from M. penetrans, M. genitalium, M. salivarium, M. pneumoniae, and M. orale also had an inhibitory effect on glucocorticoid receptor (GR) response to hormone dexamethasone (Dex) in TSU transfectants. In contrast, LAMPs from M. fermentans and M. hominis showed a stimulatory effect on the GR response to Dex in these TSU cells. The results suggest that colonization or chronic infection by mycoplasmas may significantly affect the responses of mammalian host cells to various steroid hormones, potentially affecting rates of cancer formation.

Steroid therapy attenuates acute phase reactant response among children on ventricular assist device support.

PubMed

Byrnes, Jonathan W; Bhutta, Adnan T; Rettiganti, Mallikarjuna Rao; Gomez, Alberto; Garcia, Xiomara; Dyamenahalli, Umesh; Johnson, Charles; Jaquiss, Robert D B; Imamura, Michiaki; Prodhan, Parthak

2015-04-01

Hyperfibrinogenemia, which can create a procoagulant milieu, is frequently observed in patients supported with the Berlin EXCOR (Berlin Heart GmbH, Berlin, Germany) ventricular assist device (VAD). We began initiating corticosteroids in patients with systemic inflammatory response syndrome (SIRS) episodes to mitigate hyperfibrinogenemia. We set forth to describe the impact of corticosteroids on the hyperfibrinogenemic state in our institutional experience. Retrospective data was collected on 44 consecutive patients implanted with the Berlin EXCOR VAD from April 15, 2005 through May 6, 2013. Pertinent information was abstracted from the electronic medical record. The reduction of C-reactive protein (CRP) and fibrinogen levels among days from corticosteroid treatment were described. Infections and insulin use were reported based on whether patients received steroids and if steroids were given for SIRS. Over the initial 44 Berlin EXCOR VAD implantations, 14 patients were treated with 21 courses of corticosteroids for SIRS episodes as identified by clinical features and rise in CRP. Treatment with corticosteroids reduced fibrinogen levels by day 2 to a statistically significant degree (p = 0.008). No difference in hyperglycemia or infections occurred among patients receiving corticosteroids for SIRS. Treatment with corticosteroids can potentially mitigate the SIRS response among children supported on the Berlin EXCOR VAD. In patients who received corticosteroids to mitigate inflammation, there was no increase in infections or hyperglycemia requiring insulin administration compared with patients who did not receive steroids. Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Mechanisms of protein kinase C signaling in the modulation of 3',5'-cyclic adenosine monophosphate-mediated steroidogenesis in mouse gonadal cells.

PubMed

Manna, Pulak R; Huhtaniemi, Ilpo T; Stocco, Douglas M

2009-07-01

The protein kinase C (PKC) signaling pathway plays integral roles in the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. PKC can modulate the activity of cAMP/protein kinase A signaling involved in steroidogenesis; however, its mechanism remains obscure. In the present study, we demonstrate that activation of the PKC pathway, by phorbol 12-myristate 13-acetate (PMA), was capable of potentiating dibutyryl cAMP [(Bu)(2)cAMP]-stimulated StAR expression, StAR phosphorylation, and progesterone synthesis in both mouse Leydig (MA-10) and granulosa (KK-1) tumor cells. The steroidogenic potential of PMA and (Bu)(2)cAMP was linked with phosphorylation of ERK 1/2; however, inhibition of the latter demonstrated varying effects on steroidogenesis. Transcriptional activation of the StAR gene by PMA and (Bu)(2)cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of the cAMP response element binding protein (CREB). An oligonucleotide probe containing a CREB/activating transcription factor binding region in the StAR promoter was found to bind nuclear proteins in PMA and (Bu)(2)cAMP-treated MA-10 and KK-1 cells. Chromatin immunoprecipitation studies revealed that the induction of phosphorylated CREB was tightly correlated with in vivo protein-DNA interactions and recruitment of CREB binding protein to the StAR promoter. Ectopic expression of CREB binding protein enhanced CREB-mediated transcription of the StAR gene, an event that was markedly repressed by the adenovirus E1A oncoprotein. Further studies demonstrated that the activation of StAR expression and steroid synthesis by PMA and (Bu)(2)cAMP was associated with expression of the nuclear receptor Nur77, indicating its essential role in hormone-regulated steroidogenesis. Collectively, these findings provide insight into the mechanisms by which PKC modulates cAMP/protein kinase A responsiveness involved in regulating the steroidogenic response in mouse gonadal cells.

Concentrations of the adrenocorticotropic hormone, corticosterone and sex steroid hormones and the expression of the androgen receptor in the pituitary and adrenal glands of male turkeys (Meleagris gallopavo) during growth and development.

PubMed

Kiezun, J; Kaminska, B; Jankowski, J; Dusza, L

2015-01-01

Androgens take part in the regulation of puberty and promote growth and development. They play their biological role by binding to a specific androgen receptor (AR). The aim of this study was to evaluate the expression of AR mRNA and protein in the pituitary and adrenal glands, to localize AR protein in luteinizing hormone (LH)-producing pituitary and adrenocortical cells, to determine plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone and the concentrations of corticosterone, testosterone (T), androstenedione (A4) and oestradiol (E2) in the adrenal glands of male turkeys at the age of 4, 8, 12, 16, 20, 24 and 28weeks. The concentrations of hormones and the expression of AR varied during development. The expression of AR mRNA and protein in pituitary increased during the growth. The increase of AR mRNA levels in pituitary occurred earlier than increase of AR protein. The percentage of pituitary cells expressing ARs in the population of LH-secreting cells increased in week 20. It suggests that AR expression in LH-producing pituitary cells is determined by the phase of development. The drop in adrenal AR mRNA and protein expression was accompanied by an increase in the concentrations of adrenal androgens. Those results could point to the presence of a compensatory mechanism that enables turkeys to avoid the potentially detrimental effects of high androgen concentrations. Our results will expand our knowledge of the role of steroids in the development of the reproductive system of turkeys from the first month of age until maturity. Copyright © 2015 Elsevier Inc. All rights reserved.

Androgen receptor stimulates bone sialoprotein (BSP) gene transcription via cAMP response element and activator protein 1/glucocorticoid response elements.

PubMed

Takai, Hideki; Nakayama, Youhei; Kim, Dong-Soon; Arai, Masato; Araki, Shouta; Mezawa, Masaru; Nakajima, Yu; Kato, Naoko; Masunaga, Hiroshi; Ogata, Yorimasa

2007-09-01

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. Androgens are steroid hormones that are essential for skeletal development. The androgen receptor (AR) is a transcription factor and a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. To determine the molecular mechanism involved in the stimulation of bone formation, we have analyzed the effects of androgens and AR effects on BSP gene transcription. AR protein levels were increased after AR overexpression in ROS17/2.8 cells. BSP mRNA levels were increased by AR overexpression. However, the endogenous and overexpressed BSP mRNA levels were not changed by DHT (10(-8) M, 24 h). Whereas luciferase (LUC) activities in all constructs, including a short construct (nts -116 to +60), were increased by AR overexpression, the basal and LUC activities enhanced by AR overexpression were not induced by DHT (10(-8)M, 24 h). The effect of AR overexpression was abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that AR overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE-protein complexes were supershifted by phospho-CREB antibody, and CREB, c-Fos, c-Jun, and AR antibodies disrupted the complexes formation. The AP1/GRE-protein complexes were supershifted by c-Fos antibody and c-Jun, and AR antibodies disrupted the complexes formation. These studies demonstrate that AR stimulates BSP gene transcription by targeting the CRE and AP1/GRE elements in the promoter of the rat BSP gene.

Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors.

PubMed

Zhang, Lei; Hapon, Maria B; Goyeneche, Alicia A; Srinivasan, Rekha; Gamarra-Luques, Carlos D; Callegari, Eduardo A; Drappeau, Donis D; Terpstra, Erin J; Pan, Bo; Knapp, Jennifer R; Chien, Jeremy; Wang, Xuejun; Eyster, Kathleen M; Telleria, Carlos M

2016-08-01

The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic-lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

miR-137 Targets p160 Steroid Receptor Coactivators SRC1, SRC2, and SRC3 and Inhibits Cell Proliferation

PubMed Central

Eedunuri, Vijay Kumar; Rajapakshe, Kimal; Fiskus, Warren; Geng, Chuandong; Chew, Sue Anne; Foley, Christopher; Shah, Shrijal S.; Shou, John; Mohamed, Junaith S.; O'Malley, Bert W.

2015-01-01

The p160 family of steroid receptor coactivators (SRCs) are pleiotropic transcription factor coactivators and “master regulators” of gene expression that promote cancer cell proliferation, survival, metabolism, migration, invasion, and metastasis. Cancers with high p160 SRC expression exhibit poor clinical outcomes and resistance to therapy, highlighting the SRCs as critical oncogenic drivers and, thus, therapeutic targets. microRNAs are important epigenetic regulators of protein expression. To examine the regulation of p160 SRCs by microRNAs, we used and combined 4 prediction algorithms to identify microRNAs that could target SRC1, SRC2, and SRC3 expression. For validation of these predictions, we assessed p160 SRC protein expression and cell viability after transfection of corresponding microRNA mimetics in breast cancer, uveal melanoma, and prostate cancer (PC) cell lines. Transfection of selected microRNA mimetics into breast cancer, uveal melanoma, and PC cells depleted SRC protein expression levels and exerted potent antiproliferative activity in these cell types. In particular, microRNA-137 (miR-137) depleted expression of SRC1, SRC2, and very potently, SRC3. The latter effect can be attributed to the presence of 3 miR-137 recognition sequences within the SRC3 3′-untranslated region. Using reverse phase protein array analysis, we identified a network of proteins, in addition to SRC3, that were modulated by miR-137 in PC cells. We also found that miR-137 and its host gene are epigenetically silenced in human cancer specimens and cell lines. These results support the development and testing of microRNA-based therapies (in particular based on restoring miR-137 levels) for targeting the oncogenic family of p160 SRCs in cancer. PMID:26066330

Anabolic effect of plant brassinosteroid

PubMed Central

Esposito, Debora; Komarnytsky, Slavko; Shapses, Sue; Raskin, Ilya

2011-01-01

Brassinosteroids are plant-derived polyhydroxylated derivatives of 5a-cholestane, structurally similar to cholesterol-derived animal steroid hormones and insect ecdysteroids, with no known function in mammals. 28-Homobrassinolide (HB), a steroidal lactone with potent plant growth-promoting property, stimulated protein synthesis and inhibited protein degradation in L6 rat skeletal muscle cells (EC50 4 μM) mediated in part by PI3K/Akt signaling pathway. Oral administration of HB (20 or 60 mg/kg/d for 24 d) to healthy rats fed normal diet (protein content 23.9%) increased food intake, body weight gain, lean body mass, and gastrocnemius muscle mass as compared with vehicle-treated controls. The effect of HB administration increased slightly in animals fed a high-protein diet (protein content 39.4%). Both oral (up to 60 mg/kg) and subcutaneous (up to 4 mg/kg) administration of HB showed low androgenic activity when tested in the Hershberger assay. Moreover, HB showed no direct binding to the androgen receptor in vitro. HB treatment was also associated with an improved physical fitness of untrained healthy rats, as evident from a 6.7% increase in lower extremity strength, measured by grip test. In the gastrocnemius muscle of castrated animals, HB treatment significantly increased the number of type IIa and IIb fibers and the cross-sectional area of type I and type IIa fibers. These findings suggest that oral application of HB triggers selective anabolic response with minimal or no androgenic side-effects and begin to elucidate the putative cellular targets for plant brassinosteroids in mammals.—Esposito, D., Komarnytsky, S., Shapses, S., Raskin, I. Anabolic effect of plant brassinosteroid. PMID:21746867

Lithocholic acid is an endogenous inhibitor of MDM4 and MDM2

PubMed Central

Vogel, Simon M.; Bauer, Matthias R.; Joerger, Andreas C.; Wilcken, Rainer; Brandt, Tobias; Veprintsev, Dmitry B.; Rutherford, Trevor J.; Fersht, Alan R.; Boeckler, Frank M.

2012-01-01

The proteins MDM2 and MDM4 are key negative regulators of the tumor suppressor protein p53, which are frequently upregulated in cancer cells. They inhibit the transactivation activity of p53 by binding separately or in concert to its transactivation domain. MDM2 is also a ubiquitin ligase that leads to the degradation of p53. Accordingly, MDM2 and MDM4 are important targets for drugs to inhibit their binding to p53. We found from in silico screening and confirmed by experiment that lithocholic acid (LCA) binds to the p53 binding sites of both MDM2 and MDM4 with a fivefold preference for MDM4. LCA is an endogenous steroidal bile acid, variously reported to have both carcinogenic and apoptotic activities. The comparison of LCA effects on apoptosis in HCT116 p53+/+ vs. p53-/- cells shows a predominantly p53-mediated induction of caspase-3/7. The dissociation constants are in the μM region, but only modest inhibition of binding of MDM2 and MDM4 is required to negate their upregulation because they have to compete with transcriptional coactivator p300 for binding to p53. Binding was weakened by structural changes in LCA, and so it may be a natural ligand of MDM2 and MDM4, raising the possibility that MDM proteins may be sensors for specific steroids. PMID:23035244

Dietary Fat, Fiber, and Carbohydrate Intake and Endogenous Hormone Levels in Premenopausal Women

PubMed Central

Cui, Xiaohui; Rosner, Bernard; Willett, Walter C; Hankinson, Susan E

2011-01-01

The authors conducted a cross-sectional study to investigate the associations of fat, fiber and carbohydrate intake with endogenous estrogen, androgen, and insulin-like growth factor (IGF) levels among 595 premenopausal women. Overall, no significant associations were found between dietary intake of these macronutrients and plasma sex steroid hormone levels. Dietary fat intake was inversely associated with IGF-I and IGF-binding protein 3 (IGFBP-3) levels. When substituting 5% of energy from total fat for the equivalent amount of energy from carbohydrate or protein intake, the plasma levels of IGF-I and IGFBP-3 were 2.8% (95% confidence interval [CI] 0.3, 5.3) and 1.6% (95% CI 0.4, 2.8) lower, respectively. Animal fat, saturated fat and monounsaturated fat intakes also were inversely associated with IGFBP-3 levels (P < 0.05). Carbohydrates were positively associated with plasma IGF-I level. When substituting 5% of energy from carbohydrates for the equivalent amount of energy from fat or protein intake, the plasma IGF-I level was 2.0% (95% CI 0.1, 3.9%) higher. No independent associations between fiber intake and hormone levels were observed. The results suggest that a low-fat/high-fiber or carbohydrate diet is not associated with endogenous levels of sex steroid hormones, but it may modestly increase IGF-I and IGFBP-3 levels among premenopausal women. PMID:21761370

Differential expression of steroidogenic factors 1 and 2, cytochrome p450scc, and steroidogenic acute regulatory protein in human pancreas.

PubMed

Morales, Angélica; Vilchis, Felipe; Chávez, Bertha; Morimoto, Sumiko; Chan, Carlos; Robles-Díaz, Guillermo; Díaz-Sánchez, Vicente

2008-08-01

The aim of this study was to investigate the expression of the 4 gene transcripts, steroidogenic factors 1 (SF-1) and 2 (SF-2), steroidogenic acute regulatory (StAR), and cytochrome P450 11A1, involved in the synthesis of steroid hormones in normal human pancreas. Total RNA was extracted from normal male (n = 5) and female (n = 5) samples, obtained from the organ donor program. The expression levels of SF-1, SF-2, StAR protein, and P450scc were assessed by reverse transcription-polymerase chain reaction and complemented with immunohistochemistry analysis. Polymerase chain reaction products amplification for all genes was present in both male and female samples, although differential expression was observed. The signals detected were much more evident in male than in female messenger RNA isolates for SF-1, SF-2, and StAR protein. The expression for P450scc was more intense in female samples. A similar pattern was observed in the immunohistochemical studies. Normal human pancreas expresses 4 gene transcripts involved in steroid synthesis similarly to steroidogenic organs. A distinctive characteristic is the sexually dimorphic expression of these factors. These data provide further evidence to support that the pancreas is a truly steroidogenic tissue, highlighting the presence of sex- and location-related differences in the expression of steroidogenic factors.

Early steps in steroidogenesis: intracellular cholesterol trafficking

PubMed Central

Miller, Walter L.; Bose, Himangshu S.

2011-01-01

Steroid hormones are made from cholesterol, primarily derived from lipoproteins that enter cells via receptor-mediated endocytosis. In endo-lysosomes, cholesterol is released from cholesterol esters by lysosomal acid lipase (LAL; disordered in Wolman disease) and exported via Niemann-Pick type C (NPC) proteins (disordered in NPC disease). These diseases are characterized by accumulated cholesterol and cholesterol esters in most cell types. Mechanisms for trans-cytoplasmic cholesterol transport, membrane insertion, and retrieval from membranes are less clear. Cholesterol esters and “free” cholesterol are enzymatically interconverted in lipid droplets. Cholesterol transport to the cholesterol-poor outer mitochondrial membrane (OMM) appears to involve cholesterol transport proteins. Cytochrome P450scc (CYP11A1) then initiates steroidogenesis by converting cholesterol to pregnenolone on the inner mitochondrial membrane (IMM). Acute steroidogenic responses are regulated by cholesterol delivery from OMM to IMM, triggered by the steroidogenic acute regulatory protein (StAR). Chronic steroidogenic capacity is determined by CYP11A1 gene transcription. StAR mutations cause congenital lipoid adrenal hyperplasia, with absent steroidogenesis, potentially lethal salt loss, and 46,XY sex reversal. StAR mutations initially destroy most, but not all steroidogenesis; low levels of StAR-independent steroidogenesis are lost later due to cellular damage, explaining the clinical findings. Rare P450scc mutations cause a similar syndrome. This review addresses these early steps in steroid biosynthesis. PMID:21976778

Can Supersaturation Affect Protein Crystal Quality?

NASA Technical Reports Server (NTRS)

Gorti, Sridhar

2013-01-01

In quiescent environments (microgravity, capillary tubes, gels) formation of a depletion zone is to be expected, due either to limited sedimentation, density driven convection or a combination of both. The formation of a depletion zone can: Modify solution supersaturation near crystal; Give rise to impurity partitioning. It is conjectured that both supersaturation and impurity partitioning affect protein crystal quality and size. Further detailed investigations on various proteins are needed to assess above hypothesis.

Spliceosome Protein (SRp) Regulation of Glucocorticoid Receptor Isoforms and Glucocorticoid Response in Human Trabecular Meshwork Cells

PubMed Central

Jain, Ankur; Wordinger, Robert J.; Yorio, Thomas; Clark, Abbot F.

2012-01-01

Purpose. Glaucoma is a leading cause of visual impairment and blindness, with elevated intraocular pressure (IOP) as a major causative risk factor. Glucocorticoid (GC) therapy causes morphologic and biochemical changes in the trabecular meshwork (TM), an ocular tissue involved in regulating IOP, which can lead to the development of glaucoma in susceptible individuals (steroid responders). Steroid responders comprise 40% of the general population and are at higher risk of developing glaucoma. In addition, a majority of glaucoma patients are steroid responders. Differential distribution of various isoforms of GC receptor (GR) may be responsible for this heterogeneity in the steroid response. The alternatively spliced GRβ isoform acts as dominant negative regulator of classical GRα transcriptional activity. mRNA splicing is mediated by spliceosomes, which include serine-arginine rich proteins (SRps). The purpose of this study was to determine whether specific SRps regulate levels of these isoforms and thereby GC response in TM cells. Methods. Quantitative RT-PCR, Western blot analysis, and immunocytochemistry were used to determine the differential expression of different SRps (SRp20, 30c, and 40) in human normal and glaucomatous TM cell strains. Bioinformatics was used to find putative binding sites for SRp20 and SRp40 on exon 9 of the GR gene. A peptide modulator of splicing (bombesin) and SRp expression vectors were used to modulate SRp levels and determine their effects on GRα/GRβ ratios as well as dexamethasone (DEX) responsiveness via GRE- luciferase reporter activity, fibronectin, and myocilin induction in TM cells. Results. SRp20, SRp30c, and SRp40 regulate GR splicing and the GC response in TM cells. Modulation of SRp levels altered the GRβ/α ratio that correlated with DEX responsiveness. Bombesin decreased SRp20; increased SRp30c, SRp40 levels, and GRβ/α ratio, and suppressed DEX response in TM cells. Conclusions. Relative levels of SRp20, SRp30c, and SRp40 in TM cells control differential expression of the two alternatively spliced isoforms of the GR and thereby regulate GC responsiveness. Different levels and/or activities of these SRps may account for differential GC sensitivity among the normal and glaucoma populations. PMID:22205602

Decreased serum vitamin D levels in children with asthma are associated with increased corticosteroid use.

PubMed

Searing, Daniel A; Zhang, Yong; Murphy, James R; Hauk, Pia J; Goleva, Elena; Leung, Donald Y M

2010-05-01

There is little knowledge about clinical variables associated with vitamin D (VitD) insufficiency in asthmatic children. We sought to investigate disease variables associated with VitD insufficiency in patients with childhood asthma and interaction of VitD with corticosteroid-mediated anti-inflammatory responses. We analyzed 25-hydroxyvitamin D serum levels in 100 asthmatic children to investigate relationships between 25-hydroxyvitamin D levels and patients' characteristics. We determined VitD's effects on dexamethasone (DEX) induction of mitogen-activated protein kinase phosphatase 1 and IL-10 in PBMCs. The median 25-hydroxyvitamin D serum level was 31 ng/mL. Forty-seven percent of subjects had VitD levels in the insufficient range (<30 ng/mL), whereas 17% were VitD deficient (<20 ng/mL). Log(10) IgE (P = .01, rho = -0.25) and the number of positive aeroallergen skin prick test responses (P = .02, rho = -0.23) showed a significant inverse correlation with VitD levels, whereas FEV(1) percent predicted (P = .004, rho = 0.34) and FEV(1)/forced vital capacity ratio (P = .01, rho = 0.30) showed a significant positive correlation with VitD levels. The use of inhaled steroids (P = .0475), use of oral steroids (P = .02), and total steroid dose (P = .001) all showed significant inverse correlations with VitD levels. The amount of mitogen-activated protein kinase phosphatase 1 and IL10 mRNA induced by VitD plus DEX was significantly greater than that induced by DEX alone (P < .01). In an experimental model of steroid resistance in which DEX alone did not inhibit T-cell proliferation, addition of VitD to DEX resulted in significant dose-dependent suppression of cell proliferation. Corticosteroid use and worsening airflow limitation are associated with lower VitD serum levels in asthmatic patients. VitD enhances glucocorticoid action in PBMCs from asthmatic patients and enhances the immunosuppressive function of DEX in vitro. Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Application of cellular mechanisms to growth and development of food producing animals.

PubMed

Chung, K Y; Johnson, B J

2008-04-01

Postnatal skeletal muscle growth is a result of hypertrophy of existing skeletal muscle fibers in food producing animals. Accumulation of additional nuclei, as a source of DNA, to the multinucleated skeletal muscle fiber aids in fiber hypertrophy during periods of rapid skeletal muscle growth. Muscle satellite cells are recognized as the source of nuclei to support muscle hypertrophy. Exogenous growth-enhancing compounds have been used to modulate growth rate and efficiency in meat animals for over a half century. In cattle, these compounds enhance efficiency of growth by preferentially stimulating skeletal muscle growth compared with adipose tissue. There are 2 main classes of compounds approved for use in cattle in the United States, anabolic steroids and beta-adrenergic agonists (beta-AA). Administration of both trenbolone acetate and estradiol-17beta, as implants, increased carcass protein accumulation 8 to 10% in yearling steers. Muscle satellite cells isolated from steers implanted with trenbolone acetate/ estradiol-17beta had a shorter lag phase in culture compared with satellite cells isolated from control steers. Collectively, these data indicate that activation, increased proliferation, and subsequent fusion of satellite cells in muscles of implanted cattle may be an important mechanism by which anabolic steroids enhance muscle hypertrophy. Oral administration of beta-AA to ruminants does not alter DNA accumulation in skeletal muscle over a typical feeding period (28 to 42 d). Enhanced muscle hypertrophy observed due to beta-AA feeding occurs by direct, receptor-mediated changes in protein synthesis and degradation rates of skeletal muscle tissue. Proper timing of anabolic steroid administration when coupled with beta-AA feeding could result in a synergistic response in skeletal muscle growth due to the effects of anabolic steroids at increasing satellite cell activity, which then can support the rapid hypertrophic changes of the muscle fiber when exposed to beta-AA. At the same time each of these classes of compounds are stimulating lean tissue deposition, they appear to repress adipogenesis in meat animals. Increased knowledge of the mechanism by which growth promoters regulate lean tissue deposition and adipogenesis in meat animals will allow for effective application of these techniques to optimize lean tissue growth and minimize the negative effects on meat quality.

Identification of latexin by a proteomic analysis in rat normal articular cartilage

PubMed Central

2010-01-01

Background Osteoarthritis (OA) is characterized by degeneration of articular cartilage. Animal models of OA induced are a widely used tool in the study of the pathogenesis of disease. Several proteomic techniques for selective extraction of proteins have provided protein profiles of chondrocytes and secretory patterns in normal and osteoarthritic cartilage, including the discovery of new and promising biomarkers. In this proteomic analysis to study several proteins from rat normal articular cartilage, two-dimensional electrophoresis and mass spectrometry (MS) were used. Interestingly, latexin (LXN) was found. Using an immunohistochemical technique, it was possible to determine its localization within the chondrocytes from normal and osteoarthritic articular cartilage. Results In this study, 147 proteins were visualized, and 47 proteins were identified by MS. A significant proportion of proteins are involved in metabolic processes and energy (32%), as well as participating in different biological functions including structural organization (19%), signal transduction and molecular signaling (11%), redox homeostasis (9%), transcription and protein synthesis (6%), and transport (6%). The identified proteins were assigned to one or more subcellular compartments. Among the identified proteins, we found some proteins already recognized in other studies such as OA-associated proteins. Interestingly, we identified LXN, an inhibitor of mammalian carboxypeptidases, which had not been described in articular cartilage. Immunolabeling assays for LXN showed a granular distribution pattern in the cytoplasm of most chondrocytes of the middle, deep and calcified zones of normal articular cartilage as well as in subchondral bone. In osteoarthritic cartilage, LXN was observed in superficial and deep zones. Conclusions This study provides the first proteomic analysis of normal articular cartilage of rat. We identified LXN, whose location was demonstrated by immunolabeling in the chondrocytes from the middle, deep and calcified zones of normal articular cartilage, and superficial and deep zones of osteoarthritic cartilage. PMID:20525390

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

PubMed Central

Nishimune, Hiroshi

2013-01-01

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses. PMID:22135013

Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mai, Sanyue; Qu, Xiuhua; Li, Ping

RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl wasmore » shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.« less

Elimination diets in the management of eosinophilic esophagitis.

PubMed

Wechsler, Joshua B; Schwartz, Sally; Amsden, Katie; Kagalwalla, Amir F

2014-01-01

Eosinophilic esophagitis, an increasingly recognized chronic inflammatory disorder isolated to the esophagus, is triggered by an abnormal allergic response to dietary antigens. Current treatment includes swallowed topical steroids and dietary modification, which aim to resolve symptoms and prevent long-term complications such as formation of strictures. The dietary approach has become more widely accepted because long-term steroid therapy is associated with potential risks. Dietary treatment includes elemental and elimination diets. An exclusive elemental diet, which requires replacement of all intact protein with amino acid-based formula, offers the best response of all available therapies, with remission in up to 96% of subjects proving it to be superior to all other available therapies including topical steroids. However, compliance with this approach is challenging because of poor taste and monotony. The high cost of formula and the associated psychosocial problems are additional drawbacks of this approach. Empiric and allergy test-directed elimination diets have gained popularity given that elimination of a limited number of foods is much easier and as such is more readily acceptable. There is a growing body of literature supporting this type of therapy in both children and adults. This paper reviews the evidence for all types of dietary therapy in eosinophilic esophagitis.

DNA binding triggers tetramerization of the glucocorticoid receptor in live cells

PubMed Central

Presman, Diego M.; Ganguly, Sourav; Schiltz, R. Louis; Johnson, Thomas A.; Karpova, Tatiana S.; Hager, Gordon L.

2016-01-01

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand-regulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR’s oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors. PMID:27382178

Proteasome Failure Promotes Positioning of Lysosomes around the Aggresome via Local Block of Microtubule-Dependent Transport

PubMed Central

Zaarur, Nava; Meriin, Anatoli B.; Bejarano, Eloy; Xu, Xiaobin; Gabai, Vladimir L.; Cuervo, Ana Maria

2014-01-01

Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species. PMID:24469403

Feeding an enhanced diet to Holstein heifers during the preweaning period alters steroid receptor expression and increases cellular proliferation.

PubMed

Geiger, A J; Parsons, C L M; Akers, R M

2017-10-01

Preweaning diet and estradiol treatment alters mammary development. Our objectives were to study the effects of diet and estradiol on proliferation of mammary epithelial cells and expression of estrogen receptor α (ESR1) and progesterone receptors (PGR) in these cells. Thirty-six Holstein heifer calves were raised on (1) a control milk replacer fed at 0.44 kg of powder/head per day, dry matter (DM) basis (restricted, R; 20.9% crude protein, 19.8% fat, DM basis), or (2) an enhanced milk replacer fed at 1.08 kg of powder/head per day, DM basis (Enhanced, EH; 28.9% crude protein, 26.2% fat, DM basis). Milk replacer was fed for 8 wk. At weaning, a subset (n = 6/diet) of calves were euthanized and had tissue harvested. Remaining calves received estradiol implants (E 2 ) or placebo and were euthanized at wk 10 to harvest tissue. Treatments were (1) R, (2) R + E 2 (R-E2), (3) EH, and (4) EH + E 2 (EH-E2). One day before euthanasia calves were given bromo-2'-deoxyuridine (BrdU; 5 mg/kg of body weight). At euthanization, mammary parenchyma was removed and fixed. Tissue sections from zone 1 (cisternal), 2 (medial), and 3 (distal) within the mammary gland were stained with hematoxylin and eosin and antibodies to measure expression of ESR1, PGR, and incorporation of BrdU. At wk 8, R-fed calves had more PGR-expressing cells in distal parenchyma; however, PGR expression intensity was greater in EH-fed calves. The proportion of cells expressing ESR1 was not affected by diet, but expression intensity (receptors per positive cell) was greater in EH-fed calves across all zones (62-81%). Overall, the percent BrdU-positive epithelial cells was 2 and 0.5 fold greater for EH-fed calves in zone 2 and 3. The proportion of labeled cells was greater in terminal ductal units than in subtending ducts, and treatment effects were more evident in terminal ductal units. At wk 10, calves treated with estradiol had 3.9-fold greater PGR expression intensity. The intensity and percent of cells expressing ESR1 was lowest in estradiol-treated calves. Overall, estradiol-treated calves had the greatest number of proliferating epithelial cells. Moreover, in zone 3, EH-E2 calves had a higher percentage of proliferating cells than in all other treatments. Results indicate both diet and estradiol administration alter proliferation rates of the mammary epithelium and that changes in expression of ESR1 and PGR are involved in enhanced mammary development. The data support our hypothesis that enhanced preweaning feeding increases the mammary tissue responsiveness to mammogenic stimulation. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

RBFOX2 protein domains and cellular activities.

PubMed

Arya, Anurada D; Wilson, David I; Baralle, Diana; Raponi, Michaela

2014-08-01

RBFOX2 (RNA-binding protein, Fox-1 homologue 2)/RBM9 (RNA-binding-motif protein 9)/RTA (repressor of tamoxifen action)/HNRBP2 (hexaribonucleotide-binding protein 2) encodes an RNA-binding protein involved in tissue specific alternative splicing regulation and steroid receptors transcriptional activity. Its ability to regulate specific splicing profiles depending on context has been related to different expression levels of the RBFOX2 protein itself and that of other splicing regulatory proteins involved in the shared modulation of specific genes splicing. However, this cannot be the sole explanation as to why RBFOX2 plays a widespread role in numerous cellular mechanisms from development to cell survival dependent on cell/tissue type. RBFOX2 isoforms with altered protein domains exist. In the present article, we describe the main RBFOX2 protein domains, their importance in the context of splicing and transcriptional regulation and we propose that RBFOX2 isoform distribution may play a fundamental role in RBFOX2-specific cellular effects.

Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone

PubMed Central

Wenke, Jamie L.; McDonald, W. Hayes; Schey, Kevin L.

2016-01-01

Purpose To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics. Methods Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards. Results Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ. Conclusions Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ. PMID:27537260

Cytoplasmic Localization of WT1 and Decrease of miRNA-16-1 in Nephrotic Syndrome

PubMed Central

Rangel-Ochoa, Gloria Azucena; Rodríguez-Padilla, Cristina

2017-01-01

Nephrotic syndrome (NS) is a glomerular disease that is defined by the leakage of protein into the urine and is associated with hypoalbuminemia, hyperlipidemia, and edema. Steroid-resistant NS (SRNS) patients do not respond to treatment with corticosteroids and show decreased Wilms tumor 1 (WT1) expression in podocytes. Downregulation of WT1 has been shown to be affected by certain microRNAs (miRNAs). Twenty-one patients with idiopathic NS (68.75% were SSNS and 31.25% SRNS) and 10 healthy controls were enrolled in the study. Podocyte number and WT1 location were determined by immunofluorescence, and the serum levels of miR-15a, miR-16-1, and miR-193a were quantified by RT-qPCR. Low expression and delocalization of WT1 protein from the nucleus to the cytoplasm were found in kidney biopsies of patients with SRNS and both nuclear and cytoplasmic localization were found in steroid-sensitive NS (SSNS) patients. In sera from NS patients, low expression levels of miR-15a and miR-16-1 were found compared with healthy controls, but only the miR-16-1 expression levels showed statistically significant decrease (p = 0.019). The miR-193a expression levels only slightly increased in NS patients. We concluded that low expression and delocalization from the WT1 protein in NS patients contribute to loss of podocytes while modulation from WT1 protein is not associated with the miRNAs analyzed in sera from the patients. PMID:28299339

Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine.

PubMed Central

White, R; Sjöberg, M; Kalkhoven, E; Parker, M G

1997-01-01

The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription. PMID:9135157

Mutations in COQ8B (ADCK4) found in patients with steroid-resistant nephrotic syndrome alter COQ8B function.

PubMed

Vazquez Fonseca, Luis; Doimo, Mara; Calderan, Cristina; Desbats, Maria Andrea; Acosta, Manuel J; Cerqua, Cristina; Cassina, Matteo; Ashraf, Shazia; Hildebrandt, Friedhelm; Sartori, Geppo; Navas, Placido; Trevisson, Eva; Salviati, Leonardo

2018-03-01

Mutations in COQ8B cause steroid-resistant nephrotic syndrome with variable neurological involvement. In yeast, COQ8 encodes a protein required for coenzyme Q (CoQ) biosynthesis, whose precise role is not clear. Humans harbor two paralog genes: COQ8A and COQ8B (previously termed ADCK3 and ADCK4). We have found that COQ8B is a mitochondrial matrix protein peripherally associated with the inner membrane. COQ8B can complement a ΔCOQ8 yeast strain when its mitochondrial targeting sequence (MTS) is replaced by a yeast MTS. This model was employed to validate COQ8B mutations, and to establish genotype-phenotype correlations. All mutations affected respiratory growth, but there was no correlation between mutation type and the severity of the phenotype. In fact, contrary to the case of COQ2, where residual CoQ biosynthesis correlates with clinical severity, patients harboring hypomorphic COQ8B alleles did not display a different phenotype compared with those with null mutations. These data also suggest that the system is redundant, and that other proteins (probably COQ8A) may partially compensate for the absence of COQ8B. Finally, a COQ8B polymorphism, present in 50% of the European population (NM_024876.3:c.521A > G, p.His174Arg), affects stability of the protein and could represent a risk factor for secondary CoQ deficiencies or for other complex traits. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

Effect of the NBD-group position on interaction of fluorescently-labeled cholesterol analogues with human steroidogenic acute regulatory protein STARD1.

PubMed

Tugaeva, Kristina V; Faletrov, Yaroslav V; Allakhverdiev, Elvin S; Shkumatov, Vladimir M; Maksimov, Eugene G; Sluchanko, Nikolai N

2018-02-26

Steroidogenic acute regulatory protein (StAR, STARD1) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARD1 complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARD1 interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future. Copyright © 2018 Elsevier Inc. All rights reserved.

High Expression of Galectin-3 in Patients with IgG4-Related Disease: A Proteomic Approach.

PubMed

Salah, Adeeb; Yoshifuji, Hajime; Ito, Shinji; Kitagori, Koji; Kiso, Kaori; Yamada, Norishige; Nakajima, Toshiki; Haga, Hironori; Tsuruyama, Tatsuaki; Miyagawa-Hayashino, Aya

2017-01-01

Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan condition manifesting itself in different forms. This study aimed to investigate protein expression profiles and to find the possible biomarker for IgG4-RD by liquid chromatography mass spectrometry (LC-MS) using tissue sections in IgG4-RD patients. Protein expression profiles in five IgG4-related pancreatitis and three normal pancreatic samples were compared using LC-MS and were validated by quantitative real-time PCR (qRT-PCR), immunoblotting, and immunohistochemistry. ELISA was employed in the serum of 20 patients with systemic IgG4-RD before and during steroid treatment. LC-MS indicated that the levels of 17 proteins were significantly higher and 12 others were significantly lower in IgG4-related pancreatitis patients compared to controls. Among these proteins, galectin-3 levels were 13-fold higher in IgG4-related pancreatitis ( P < 0.01). These results were confirmed by immunoblotting and qRT-PCR. The average number of galectin-3 + cells in various organs of IgG4-RD patients, including salivary glands, lungs, and lymph nodes, was higher than in controls. Galectin-3 was detectable in macrophages, dendritic cells, and stromal myofibroblast-like cells, but not in lymphocytes by immunofluorescence staining. Serum galectin-3 levels were higher in patients with IgG4-RD compared with healthy donors and remained high during steroid therapy. Galectin-3 was overexpressed in IgG4-RD and the levels were indirectly related to clinical activity.

High Expression of Galectin-3 in Patients with IgG4-Related Disease: A Proteomic Approach

PubMed Central

Salah, Adeeb; Yoshifuji, Hajime; Ito, Shinji; Kitagori, Koji; Kiso, Kaori; Yamada, Norishige; Nakajima, Toshiki; Haga, Hironori; Tsuruyama, Tatsuaki

2017-01-01

Objectives Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan condition manifesting itself in different forms. This study aimed to investigate protein expression profiles and to find the possible biomarker for IgG4-RD by liquid chromatography mass spectrometry (LC-MS) using tissue sections in IgG4-RD patients. Methods Protein expression profiles in five IgG4-related pancreatitis and three normal pancreatic samples were compared using LC-MS and were validated by quantitative real-time PCR (qRT-PCR), immunoblotting, and immunohistochemistry. ELISA was employed in the serum of 20 patients with systemic IgG4-RD before and during steroid treatment. Results LC-MS indicated that the levels of 17 proteins were significantly higher and 12 others were significantly lower in IgG4-related pancreatitis patients compared to controls. Among these proteins, galectin-3 levels were 13-fold higher in IgG4-related pancreatitis (P < 0.01). These results were confirmed by immunoblotting and qRT-PCR. The average number of galectin-3 + cells in various organs of IgG4-RD patients, including salivary glands, lungs, and lymph nodes, was higher than in controls. Galectin-3 was detectable in macrophages, dendritic cells, and stromal myofibroblast-like cells, but not in lymphocytes by immunofluorescence staining. Serum galectin-3 levels were higher in patients with IgG4-RD compared with healthy donors and remained high during steroid therapy. Conclusion Galectin-3 was overexpressed in IgG4-RD and the levels were indirectly related to clinical activity. PMID:28593065

Behavioral evidence for sex steroids hypersensitivity in castrated male canaries.

PubMed

Shevchouk, Olesya; Ghorbanpoor, Samar; Smith, Ed; Liere, Philippe; Schumacher, Michael; Ball, Gregory F; Cornil, Charlotte A; Balthazart, Jacques

2018-06-14

In seasonally breeding songbirds such as canaries, singing behavior is predominantly under the control of testosterone and its metabolites. Short daylenths in the fall that break photorefractoriness are followed by increasing daylengths in spring that activate singing via both photoperiodic and hormonal mechanisms. However, we observed in a group of castrated male Fife fancy canaries maintained for a long duration under a short day photoperiod a large proportion of subjects that sang at high rates. This singing rate was not correlated with variation in the low circulating concentrations of testosterone. Treatment of these actively singing castrated male canaries with a combination of an aromatase inhibitor (ATD) and an androgen receptor blocker (flutamide) only marginally decreased this singing activity as compared to control untreated birds and did not affect various measures of song quality. The volumes of HVC and of the medial preoptic nucleus (POM) were also unaffected by these treatments but were relatively large and similar to volumes in testosterone-treated males. In contrast, peripheral androgen-sensitive structures such as the cloacal protuberance and syrinx mass were small, similar to what is observed in castrates. Together these data suggest that after a long-term steroid deprivation singing behavior can be activated by very low concentrations of testosterone. Singing normally depends on the activation by testosterone and its metabolites of multiple downstream neurochemical systems such as catecholamines, nonapeptides or opioids. These transmitter systems might become hypersensitive to steroid action after long term castration as they probably are at the end of winter during the annual cycle in seasonally breeding temperate zone species. Copyright © 2017. Published by Elsevier Inc.

Assessing the links among environmental contaminants, endocrinology, and parasites to understand amphibian declines in montane regions of Costa Rica.

PubMed

Leary, Christopher J; Ralicki, Hannah F; Laurencio, David; Crocker-Buta, Sarah; Malone, John H

2018-01-01

Amphibians inhabiting montane riparian zones in the Neotropics are particularly vulnerable to decline, but the reasons are poorly understood. Because environmental contaminants, endocrine disruption, and pathogens often figure prominently in amphibian declines it is imperative that we understand how these factors are potentially interrelated to affect montane populations. One possibility is that increased precipitation associated with global warming promotes the deposition of contaminants in montane regions. Increased exposure to contaminants, in turn, potentially elicits chronic elevations in circulating stress hormones that could contribute to montane population declines by compromising resistance to pathogens and/or production of sex steroids regulating reproduction. Here, we test this hypothesis by examining contaminant levels, stress and sex steroid levels, and nematode abundances in male drab treefrogs, Smilisca sordida, from lowland and montane populations in Costa Rica. We found no evidence that montane populations were more likely to possess contaminants (i.e., organochlorine, organophosphate and carbamate pesticides or benzidine and chlorophenoxy herbicides) than lowland populations. We also found no evidence of elevational differences in circulating levels of the stress hormone corticosterone, estradiol or progesterone. However, montane populations possessed lower androgen levels, hosted more nematode species, and had higher nematode abundances than lowland populations. Although these results suggested that nematodes contributed to lower androgens in montane populations, we were unable to detect a significant inverse relationship between nematode abundance and androgen level. Our results suggest that montane populations of this species are not at greater risk of exposure to contaminants or chronic stress, but implicate nematodes and compromised sex steroid levels as potential threats to montane populations.

Mitochondrial metabolic regulation by GRP78

PubMed Central

Prasad, Manoj; Pawlak, Kevin J.; Burak, William E.; Perry, Elizabeth E.; Marshall, Brendan; Whittal, Randy M.; Bose, Himangshu S.

2017-01-01

Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity. PMID:28275724

The cis decoy against the estrogen response element suppresses breast cancer cells via target disrupting c-fos not mitogen-activated protein kinase activity.

PubMed

Wang, Li Hua; Yang, Xiao Yi; Zhang, Xiaohu; Mihalic, Kelly; Xiao, Weihua; Farrar, William L

2003-05-01

Breast cancer, the most common malignancy in women, has been demonstrated to be associated with the steroid hormone estrogen and its receptor (ER), a ligand-activated transcription factor. Therefore, we developed a phosphorothiolate cis-element decoy against the estrogen response element (ERE decoy) to target disruption of ER DNA binding and transcriptional activity. Here, we showed that the ERE decoy potently ablated the 17beta-estrogen-inducible cell proliferation and induced apoptosis of human breast carcinoma cells by functionally affecting expression of c-fos gene and AP-1 luciferase gene reporter activity. Specificity of the decoy was demonstrated by its ability to directly block ER binding to a cis-element probe and transactivation. Moreover, the decoy failed to inhibit ER-mediated mitogen-activated protein kinase signaling pathways and cell growth of ER-negative breast cancer cells. Taken together, these data suggest that estrogen-mediated cell growth of breast cancer cells can be preferentially restricted via targeted disruption of ER at the level of DNA binding by a novel and specific decoy strategy applied to steroid nuclear receptors.

Molecular identity and gene expression of aldosterone synthase cytochrome P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamoto, Mitsuhiro; Nonaka, Yasuki; Takemori, Hiroshi

11{beta}-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11{beta}-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolatedmore » from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.« less

Non-steroidal Anti-inflammatory Drugs Attenuate Hyperalgesia and Block Upregulation of Trigeminal Ganglionic Sodium Channel 1.7 after Induction of Temporomandibular Joint Inflammation in Rats.

PubMed

Bi, Rui Yun; Ding, Yun; Gan, Ye Hua

2016-03-01

To investigate the association between the analgesic effect of non-steroidal antiinflammatory drugs (NSAIDs) and sodium channel 1.7 (Nav1.7) expression in the trigeminal ganglion (TG). Temporomandibular joint (TMJ) inflammation was induced by complete Freund's adjuvant (CFA) in female rats. Ibuprofen, diclofenac sodium and meloxicam were given intragastrically before induction of TMJ inflammation. Histopathological evaluation and scoring of TMJ inflammation was used to evaluate the level of inflammation. The head withdrawal threshold and food intake were measured to evaluate TMJ nociceptive responses. The mRNA and protein expression of trigeminal ganglionic Nav1.7 was examined using real-time polymerase chain reaction and western blot. Twenty-four hours after the injection of CFA into the TMJs, NSAIDs attenuated hyperalgesia of inflamed TMJ and simultaneously blocked inflammation-induced upregulation of Nav1.7 mRNA and protein expression in the TG. However, ibuprofen and diclofenac sodium slightly attenuated TMJ inflammation and meloxicam did not affect TMJ inflammation. Attenuation of hyperalgesia of inflamed TMJ by NSAIDs might be associated with their role in blocking upregulation of trigeminal ganglionic Nav1.7.

Structural architecture of the human long non-coding RNA, steroid receptor RNA activator

PubMed Central

Novikova, Irina V.; Hennelly, Scott P.; Sanbonmatsu, Karissa Y.

2012-01-01

While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features. PMID:22362738

Proteoglycans in Leiomyoma and Normal Myometrium: Abundance, Steroid Hormone Control, and Implications for Pathophysiology.

PubMed

Barker, Nichole M; Carrino, David A; Caplan, Arnold I; Hurd, William W; Liu, James H; Tan, Huiqing; Mesiano, Sam

2016-03-01

Uterine leiomyoma are a common benign pelvic tumors composed of modified smooth muscle cells and a large amount of extracellular matrix (ECM). The proteoglycan composition of the leiomyoma ECM is thought to affect pathophysiology of the disease. To test this hypothesis, we examined the abundance (by immunoblotting) and expression (by quantitative real-time polymerase chain reaction) of the proteoglycans biglycan, decorin, and versican in leiomyoma and normal myometrium and determined whether expression is affected by steroid hormones and menstrual phase. Leiomyoma and normal myometrium were collected from women (n = 17) undergoing hysterectomy or myomectomy. In vitro studies were performed on immortalized leiomyoma (UtLM) and normal myometrial (hTERT-HM) cells with and without exposure to estradiol and progesterone. In leiomyoma tissue, abundance of decorin messenger RNA (mRNA) and protein were 2.6-fold and 1.4-fold lower, respectively, compared with normal myometrium. Abundance of versican mRNA was not different between matched samples, whereas versican protein was increased 1.8-fold in leiomyoma compared with myometrium. Decorin mRNA was 2.4-fold lower in secretory phase leiomyoma compared with proliferative phase tissue. In UtLM cells, progesterone decreased the abundance of decorin mRNA by 1.3-fold. Lower decorin expression in leiomyoma compared with myometrium may contribute to disease growth and progression. As decorin inhibits the activity of specific growth factors, its reduced level in the leiomyoma cell microenvironment may promote cell proliferation and ECM deposition. Our data suggest that decorin expression in leiomyoma is inhibited by progesterone, which may be a mechanism by which the ovarian steroids affect leiomyoma growth and disease progression. © The Author(s) 2015.

Modulation of steroidogenesis and estrogen signalling in the estuarine killifish (Fundulus heteroclitus) exposed to ethinylestradiol.

PubMed

Hogan, Natacha S; Currie, Suzanne; LeBlanc, Sacha; Hewitt, L Mark; MacLatchy, Deborah L

2010-06-10

Previous studies have shown that mummichog (Fundulus heteroclitus; a lunar, asynchronous-spawning killifish of the western Atlantic) exposed to 17alpha-ethynylestradiol (EE2) exhibit decreased plasma reproductive steroid levels, decreased gonadal steroid production, increased plasma vitellogenin, decreased fecundity and impaired fertilization. The objective of this study was to determine the potential mechanisms by which EE2 depresses gonadal steroidogenesis and influences estrogen signalling in the mummichog. Adult recrudesced fish were exposed to the potent synthetic estrogen, ethinylestradiol (EE2; 0-270ng/L) for 14 days. Following exposure, gonadal tissue was removed and incubated for 24h with stimulators of steroidogenesis, including forskolin; 25-OH cholesterol; or pregnenolone. Testosterone production was decreased in basal, forskolin-stimulated and pregnenolone-stimulated EE2-exposed males, indicating effects on the steroidogenic pathway both at and downstream of cholesterol mobilization to P450 side-chain cleavage (P450scc) and/or P450scc conversion of cholesterol to pregnenolone. Hepatic transcript levels of estrogen receptor alpha (ERalpha) and vitellogenin were increased in EE2-treated males compared to control recrudescing males and females confirming an estrogenic response. Hepatic heat shock protein 90 (Hsp90), a chaperoning molecule involved in estrogen signalling, was not affected by EE2 exposure at either the transcript or protein level. However, higher levels of Hsp90 observed in the membrane fractions of female fish raise interesting questions regarding the influence of gender on Hsp90's role in estrogen signalling. These results demonstrate that EE2 can alter steroid production at specific sites within the steroidogenic pathway and can stimulate hepatic estrogen signalling, providing important information regarding the molecular mechanisms underlying the endocrine response of the mummichog to exogenous estrogen.

Rapid Steroid Hormone Actions Initiated at the Cell Surface and the Receptors that Mediate Them with an Emphasis on Recent Progress in Fish Models

PubMed Central

Thomas, Peter

2011-01-01

In addition to the classic genomic mechanism of steroid action mediated by activation of intracellular nuclear receptors, there is now extensive evidence that steroids also activate receptors on the cell surface to initiate rapid intracellular signaling and biological responses that are often nongenomic. Recent progress in our understanding of rapid, cell surface-initiated actions of estrogens, progestins, androgens and corticosteroids and the identities of the membrane receptors that act as their intermediaries is briefly reviewed with a special emphasis on studies in teleost fish. Two recently discovered novel proteins with seven-transmembrane domains, G protein-coupled receptor 30 (GPR30), and membrane progestin receptors (mPRs) have the ligand binding and signaling characteristics of estrogen and progestin membrane receptors, respectively, but their functional significance is disputed by some researchers. GPR30 is expressed on the cell surface of fish oocytes and mediates estrogen inhibition of oocyte maturation. mPRα is also expressed on the oocyte cell surface and is the intermediary in progestin induction of oocyte maturation in fish. Recent results suggest there is cross-talk between these two hormonal pathways and that there is reciprocal down-regulation of GPR30 and mPRα expression by estrogens and progestins at different phases of oocyte development to regulate the onset of oocyte maturation. There is also evidence in fish that mPRs are involved in progestin induction of sperm hypermotility and anti-apoptotic actions in ovarian follicle cells. Nonclassical androgen and corticosteroid actions have also been described in fish models but the membrane receptors mediating these actions have not been identified. PMID:22154643

Caged mudsnail Potamopyrgus antipodarum (Gray) as an integrated field biomonitoring tool: exposure assessment and reprotoxic effects of water column contamination.

PubMed

Gust, M; Gagné, F; Berlioz-Barbier, A; Besse, J P; Buronfosse, T; Tournier, M; Tutundjian, R; Garric, J; Cren-Olivé, C

2014-05-01

This study highlights the usefulness of gastropods for water quality monitoring. Gastropods were caged upstream and downstream of an effluent discharge. Exposure was assessed by measurement of organic contaminants in water. Contamination of the Potamopyrgus antipodarum mudsnail was also measured using innovative techniques at the end of the 42 days of exposure. Biological effects were measured at the individual level (growth, reproduction) and subindividual level (energy reserves, vitellin-like proteins, steroid levels, expression of genes involved in estrogen signaling pathways), thus providing a better understanding of reprotoxic effects. The effluent was mainly contaminated by pharmaceutical compounds, as was the mudsnail. The highest concentrations were measured for oxazepam and were higher than 2 mg/kg downstream of the effluent discharge. Alkylphenols, bisphenol A, and vertebrate-like sex-steroid hormones were also bioaccumulated by the mudsnail downstream of the effluent. The combined use of water and snail contamination provided a complete exposure assessment. Exposure was further linked to biological effects. The mudsnail was shown to be a better adapted species for in situ exposures than Valvata piscinalis. Reproduction was sharply decreased after 6 weeks of exposure in the mudsnail. Feeding issues were excluded, confirming the toxic origin. These effects were related to estrogen signaling pathways using genomic analysis. Genes coding for proteins involved in nongenomic signaling pathways were inhibited, and those of genomic pathway repressors were induced. These results suggest that the chemical contamination due to the effluent discharge altered steroid control of reproduction and blocked the transition between oocyte and unshelled embryo, resulting in a drastic decrease of embryo production, while survival was not affected. Copyright © 2014 Elsevier Ltd. All rights reserved.

Endocrine disruption: In silico interactions between phthalate plasticizers and corticosteroid binding globulin.

PubMed

Sheikh, Ishfaq A; Beg, Mohd A

2017-12-01

Endocrine disruption is a phenomenon when a man-made or natural compound interferes with normal hormone function in human or animal body systems. Endocrine-disrupting compounds (EDCs) have assumed considerable importance as a result of industrial activity, mass production of synthetic chemicals and environmental pollution. Phthalate plasticizers are a group of chemicals used widely and diversely in industry especially in the plastic industry, and many of the phthalate compounds have endocrine-disrupting properties. Increasing evidence indicates that steroid nuclear receptors and steroid binding proteins are the main targets of endocrine disruption. Corticosteroid-binding globulin (CBG) is a steroid binding protein that binds and transports cortisol in the blood circulation and is a potential target for endocrine disruption. An imbalance of cortisol in the body leads to many health problems. Induced fit docking of nine important and environmentally relevant phthalate plasticizers (DMP, BBP, DBP, DIBP, DnHP, DEHP, DINP, DnOP, DIDP) showed interactions with 10-19 amino acid residues of CBG. Comparison of the interacting residues of CBG with phthalate ligands and cortisol showed an overlapping of the majority (53-82%) of residues for each phthalate. Five of nine phthalate compounds and cortisol shared a hydrogen bonding interaction with the Arg-252 residue of CBG. Long-chain phthalates, such as DEHP, DINP, DnOP and DIDP displayed a higher binding affinity and formed a number of interactions with CBG in comparison to short-chain phthalates. The similarity in structural binding characteristics of phthalate compounds and native ligand cortisol suggested potential competitive conflicts in CBG-cortisol binding function and possible disruption of cortisol and progesterone homeostasis. Copyright © 2017 John Wiley & Sons, Ltd.

Dissection of androgen receptor-promoter interactions: Steroid receptors partition their interaction energetics in parallel with their phylogenetic divergence

PubMed Central

De Angelis, Rolando W; Yang, Qin; Miura, Michael T; Bain, David L

2013-01-01

Steroid receptors comprise a homologous family of ligand-activated transcription factors. The members include androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and progesterone receptor (PR). Phylogenetic studies demonstrate that AR, GR, MR and PR are most closely related, falling into subgroup 3C. ER is more distantly related, falling into subgroup 3A. To determine the quantitative basis by which receptors generate their unique transcriptional responses, we are systematically dissecting the promoter-binding energetics of all receptors under a single “standard state” condition. Here we examine the self-assembly and promoter-binding energetics of full-length AR and a mutant associated with prostate cancer, T877A. We first demonstrate that both proteins exist only as monomers, showing no evidence of dimerization. Although this result contradicts the traditional understanding that steroid receptors dimerize in the absence of DNA, it is fully consistent with our previous work demonstrating that GR and two PR isoforms either do not dimerize or dimerize only weakly. Moreover, both AR proteins exhibit substantial cooperativity between binding sites, again as seen for GR and PR. In sharp contrast, the more distantly related ER-α dimerizes so strongly that energetics can only be measured indirectly, yet cooperativity is negligible. Thus homologous receptors partition their promoter-binding energetics quite differently. Moreover, since receptors most closely related by phylogeny partition their energetics similarly, such partitioning appears to be evolutionarily conserved. We speculate that such differences in energetics, coupled with different promoter architectures, serve as the basis for generating receptor-specific promoter occupancy and thus function. PMID:23917122

X-Ray Crystal Structure of the Ancestral 3-Ketosteroid Receptor-Progesterone-Mifepristone Complex Shows Mifepristone Bound at the Coactivator Binding Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colucci, Jennifer K.; Ortlund, Eric A.

2013-12-12

Steroid receptors are a subfamily of nuclear receptors found throughout all metazoans. They are highly important in the regulation of development, inflammation, and reproduction and their misregulation has been implicated in hormone insensitivity syndromes and cancer. Steroid binding to SRs drives a conformational change in the ligand binding domain that promotes nuclear localization and subsequent interaction with coregulator proteins to affect gene regulation. SRs are important pharmaceutical targets, yet most SR-targeting drugs have off-target pharmacology leading to unwanted side effects. A better understanding of the structural mechanisms dictating ligand specificity and the evolution of the forces that created the SR-hormonemore » pairs will enable the design of better pharmaceutical ligands. In order to investigate this relationship, we attempted to crystallize the ancestral 3-ketosteroid receptor (ancSR2) with mifepristone, a SR antagonist. Here, we present the x-ray crystal structure of the ancestral 3-keto steroid receptor (ancSR2)-progesterone complex at a resolution of 2.05 Å. This improves upon our previously reported structure of the ancSR2-progesterone complex, permitting unambiguous assignment of the ligand conformation within the binding pocket. Surprisingly, we find mifepristone, fortuitously docked at the protein surface, poised to interfere with coregulator binding. Recent attention has been given to generating pharmaceuticals that block the coregulator binding site in order to obstruct coregulator binding and achieve tissue-specific SR regulation independent of hormone binding. Mifepristone’s interaction with the coactivator cleft of this SR suggests that it may be a useful molecular scaffold for further coactivator binding inhibitor development.« less

Effects of topical steroids on tight junction proteins and spongiosis in esophageal epithelia of patients with eosinophilic esophagitis.

PubMed

Katzka, David A; Tadi, Ravikanth; Smyrk, Thomas C; Katarya, Eesha; Sharma, Anamay; Geno, Deborah M; Camilleri, Michael; Iyer, Prasad G; Alexander, Jeffrey A; Buttar, Navtej S

2014-11-01

The allergic response associated with eosinophilic esophagitis (EoE) occurs when food antigens permeate tight junction-mediated epithelial dilated intercellular spaces. We assessed whether levels of tight junction proteins correlate with the dilation of intercellular spaces (spongiosis) and the effects of topical steroids on these parameters. We assessed esophageal biopsy samples from 10 patients with active EoE treated with topical fluticasone, 10 untreated patients, and 10 patients without esophageal disease (controls) for degree of spongiosis. Immunohistochemical assays were used to determine the levels of the tight junction proteins filaggrin, zonula occludens (ZO)-1, ZO-2, ZO-3, and claudin-1. Histology and immunohistochemistry results were assessed blindly, with levels of tight junction proteins and degree of spongiosis rated on scales of 0 to 3. The mean degrees of spongiosis in untreated and treated patients with EoE were 1.3 and 0.4, respectively (P = .016). Esophageal epithelia did not stain significantly for ZO-1 or ZO-2. Filaggrin was observed in a predominant cytoplasmic pattern, compared with the cytoplasmic and membranous patterns of ZO-3 and claudin-1. In biopsy specimens from patients with active EoE, the mean staining intensities for filaggrin, ZO-3, and claudin-1 were 1.6, 1.4, and 0.7, respectively. In biopsy specimens from patients treated with fluticasone, levels of filaggrin, ZO-3, and claudin-1 were 2.8 (P = .002 compared with untreated patients), 1.7 (P = .46 compared with untreated patients), and 1.3 (P = .25 compared with untreated patients), respectively. The correlation between the level of filaggrin and the degree of spongiosis was r = 0.23, and between ZO-3 staining and the degree of spongiosis was r = .016 (P = .001 for filaggrin vs ZO-3 staining). Filaggrin, ZO-3, and claudin-1 (but not ZO-1 or ZO-2) are detected in the esophageal mucosa of patients with EoE treated with steroids and individuals without esophageal disease. Without treatment, spongiosis increases, corresponding with reduced levels of filaggrin, ZO-3, and claudin-1. Loss of tight junction regulators and dilation of intercellular spaces appear to be involved in the pathophysiology of EoE and could be targets for treatment. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

[Effects of exogenous high mobility group protein box 1 on angiogenesis in ischemic zone of early scald wounds of rats].

PubMed

Dai, L; Guo, X; Huang, H J; Liao, X M; Luo, X Q; Li, D; Zhou, H; Gao, X C; Tan, M Y

2018-04-20

Objective: To observe effects of exogenous high mobility group protein box 1 (HMGB1) on angiogenesis in ischemic zone of early scald wounds of rats. Methods: Thirty-six Sprague-Dawley rats were divided into HMGB1 group and simple scald (SS) group according to the random number table, with 18 rats in each group. Comb-like copper mould was placed on the back of rats for 20 s after being immersed in 100 ℃ hot water for 3 to 5 min to make three ischemic zones of wound. Immediately after scald, rats in HMGB1 group were subcutaneously injected with 0.4 μg HMGB1 and 0.1 mL phosphate buffer solution (PBS), and rats in SS group were subcutaneously injected with 0.1 mL PBS from boarders of ischemic zone of scald wound. At post scald hour (PSH) 24, 48, and 72, 6 rats in each group were collected. Protein expressions of vascular endothelial growth factor (VEGF) in ischemic zone of wound at PSH 24, 48, and 72 and protein expressions of CD31 in ischemic zone of wound at PSH 48 and 72 were detected by immunohistochemistry. The number of microvessel in CD31 immunohistochemical sections of ischemic zone of wound at PSH 48 and 72 was calculated after observing by the microscope. The mRNA expressions of VEGF and CD31 in ischemic zone of wound were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction at PSH 24, 48, and 72. Data were processed with analysis of variance of factorial design, t test, and Bonferroni correction. Results: (1) At PSH 24, 48, and 72, protein expressions of VEGF in ischemic zone of wound of rats in HMGB1 group were significantly higher than those of rats in SS group ( t =7.496, 4.437, 5.402, P <0.05 or P <0.01). At PSH 48 and 72, protein expressions of CD31 in ischemic zone of wound of rats in HMGB1 group were 0.038 8±0.007 9 and 0.057 7±0.001 2 respectively, significantly higher than 0.013 4±0.004 9 and 0.030 3±0.004 0 of rats in SS group ( t =10.257, 15.055, P <0.01). (2) At PSH 48 and 72, the number of microvessel in ischemic zone of wound of rats in HMGB1 group was obviously more than that of rats in SS group ( t =3.536, 4.000, P <0.05). (3) At PSH 24, 48, and 72, mRNA expressions of VEGF in ischemic zone of wound of rats in HMGB1 group were significantly higher than those of rats in SS group ( t =4.406, 3.821, 3.356, P <0.05). At PSH 24 and 48, mRNA expressions of CD31 in ischemic zone of wound of rats in HMGB1 group were significantly higher than those of rats in SS group ( t =4.113, 3.466, P <0.05). At PSH 72, mRNA expressions of CD31 in ischemic zone of wound of rats in 2 groups were close ( t =0.010, P >0.05). Conclusions: Exogenous HMGB1 can promote angiogenesis in ischemic zone of early scald wounds of rats by increasing expressions of VEGF and CD31.

Endometrial proteins: a reappraisal.

PubMed

Seppälä, M; Julkunen, M; Riittinen, L; Koistinen, R

1992-06-01

Uterine factors influence reproduction at the macro-anatomy level, and the effects of hormonal steroids on endometrial morphology are well recognized in the histopathological diagnosis of dysfunctional bleeding and infertility. During the past decade, attention has been paid to endometrial protein synthesis and secretion with respect to endocrine stimuli and implantation, and to the paracrine/autocrine effects of endometrial peptide growth factors, their binding proteins and other factors. The emphasis of this presentation is on protein secretion of the secretory endometrium, in which progesterone plays a pivotal role. Insulin-like growth factors have receptors on the endometrium, and IGF-binding proteins, stimulated by progesterone, modulate the effects of IGFs locally. Also other protein products of the secretory endometrium have been reviewed in this communication, with special emphasis on studies of a progesterone-associated endometrial protein which has many names in the literature, such as PEP, PP14, alpha 2-PEG and AUP. Extensive studies are ongoing in many laboratories to elucidate the regulation, function, interplay at tissue and cellular levels, and clinical significance of these proteins.

Active zones of mammalian neuromuscular junctions: formation, density, and aging.

PubMed

Nishimune, Hiroshi

2012-12-01

Presynaptic active zones are synaptic vesicle release sites that play essential roles in the function and pathology of mammalian neuromuscular junctions (NMJs). The molecular mechanisms of active zone organization use presynaptic voltage-dependent calcium channels (VDCCs) in NMJs as scaffolding proteins. VDCCs interact extracellularly with the muscle-derived synapse organizer, laminin β2 and interact intracellularly with active zone-specific proteins, such as Bassoon, CAST/Erc2/ELKS2alpha, ELKS, Piccolo, and RIMs. These molecular mechanisms are supported by studies in P/Q- and N-type VDCCs double-knockout mice, and they are consistent with the pathological conditions of Lambert-Eaton myasthenic syndrome and Pierson syndrome, which are caused by autoantibodies against VDCCs or by a laminin β2 mutation. During normal postnatal maturation, NMJs maintain the density of active zones, while NMJs triple their size. However, active zones become impaired during aging. Propitiously, muscle exercise ameliorates the active zone impairment in aged NMJs, which suggests the potential for therapeutic strategies. © 2012 New York Academy of Sciences.

Transcriptome Analysis of Tomato Flower Pedicel Tissues Reveals Abscission Zone-Specific Modulation of Key Meristem Activity Genes

PubMed Central

Sun, Xiuli; Zhang, Rongzhi; Wu, Liang; Liang, Yanchun; Mao, Long

2013-01-01

Tomato flower abscises at the anatomically distinct abscission zone that separates the pedicel into basal and apical portions. During abscission, cell separation occurs only at the abscission zone indicating distinctive molecular regulation in its cells. We conducted a transcriptome analysis of tomato pedicel tissues during ethylene promoted abscission. We found that the abscission zone was the most active site with the largest set of differentially expressed genes when compared with basal and apical portions. Gene Ontology analyses revealed enriched transcription regulation and hydrolase activities in the abscission zone. We also demonstrate coordinated responses of hormone and cell wall related genes. Besides, a number of ESTs representing homologs of key Arabidopsis shoot apical meristem activity genes were found to be preferentially expressed in the abscission zone, including WUSCHEL (WUS), KNAT6, LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN 1(LBD1), and BELL-like homeodomain protein 1 (BLH1), as well as tomato axillary meristem genes BLIND (Bl) and LATERAL SUPPRESSOR (Ls). More interestingly, the homologs of WUS and the potential functional partner OVATE FAMILIY PROTEIN (OFP) were subsequently down regulated during abscission while Bl and AGL12 were continuously and specifically induced in the abscission zone. The expression patterns of meristem activity genes corroborate the idea that cells of the abscission zone confer meristem-like nature and coincide with the course of abscission and post-abscission cell differentiation. Our data therefore propose a possible regulatory scheme in tomato involving meristem genes that may be required not only for the abscission zone development, but also for abscission. PMID:23390523

Pediatric Bilateral Blue Laser Pointer-Induced Maculopathy

PubMed Central

Raevis, Joseph; Shrier, Eric

2017-01-01

Background We report the first case of pediatric bilateral blue laser pointer maculopathy with complete resolution of visual symptoms. Case A 12-year-old boy presented with bilateral decreased visual acuity and central scotomata after blue laser pointer exposure. He was treated with a Medrol Dosepak and topical nonsteroidal anti-inflammatory drug (NSAID), with gradual visual acuity improved from 20/40 OU to 20/20 OU over 22 weeks, but with persistent evidence of outer retinal layer disruption from the external limiting membrane to the interdigitation zone. Conclusion Oral steroids and topical NSAIDs may be effective in improving visual outcomes in laser pointer maculopathy in the pediatric population. PMID:28611647

Influence of north climatic conditions on the peat lipids composition

NASA Astrophysics Data System (ADS)

Serebrennikova, O. V.; Strelnikova, E. B.; Duchko, M. A.; Preis, Yu I.

2018-03-01

The paper studies the composition of lipid organic compounds of peat from the northern regions of the Russian Federation. Peat was sampled in the northern taiga, forest-tundra and tundra zones, characterized by various hydrothermal conditions and vegetation cover. n-Alkanes, fatty acids and their ethers, aldehydes, ketones, alcohols, tocopherols, squalene, bi-, tri- and pentacyclic terpenoids, as well as steroids were identified in peat lipids by gas chromatography-mass spectrometry. The dependences of the total content of lipids and the majority of the investigated compounds classes on the ambient temperature and vegetation, as well as the correlation between the composition of n-alkanes and humidity were revealed.

CRMPs colocalize and interact with cytoskeleton in hippocampal neurons

PubMed Central

Yang, Yuhao; Zhao, Bo; Ji, Zhisheng; Zhang, Guowei; Zhang, Jifeng; Li, Sumei; Guo, Guoqing; Lin, Hongsheng

2015-01-01

CRMP family proteins (CRMPs) are widely expressed in the developing neurons, mediating a variety of fundamental functions such as growth cone guidance, neuronal polarity and axon elongation. However, whether all the CRMP proteins interact with cytoskeleton remains unknown. In this study, we found that in cultured hippocampal neurons, CRMPs mainly colocalized with tubulin and actin network in neurites. In growth cones, CRMPs colocalized with tubulinmainly in the central (C-) domain and transition zone (T-zone), less in the peripheral (P-) domain and colocalized with actin in all the C-domain, T-zone and P-domain. The correlation efficiency of CRMPs between actin was significantly higher than that between tubulin, especially in growth cones. We successfully constructed GST-CRMPs plasmids, expressed and purified the GST-CRMP proteins. By GST-pulldown assay, all the CRMP family proteins were found to beinteracted with cytoskeleton proteins. Taken together, we revealed that CRMPs were colocalized with cytoskeleton in hippocampal neurons, especially in growth cones. CRMPs can interact with both tubulin and actin, thus mediating neuronal development. PMID:26885211

Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone.

PubMed

Zhang, Zhe; Voothuluru, Priyamvada; Yamaguchi, Mineo; Sharp, Robert E; Peck, Scott C

2013-01-01

Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM) proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein implicates that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Anabolic Steroids...What's the Hype?...

ERIC Educational Resources Information Center

Landry, Gregory L.; Wagner, Lauris L.

This pamphlet uses a question-and-answer format to examine the use and abuse of anabolic steroids. It begins by explaining that all steroids are not anabolic steroids and that anabolic steroids are those used specifically to build muscles quickly. Medical uses of anabolic steroids are reviewed; how people get steroids, how they take them, and…

Steroid resistance in COPD is associated with impaired molecular chaperone Hsp90 expression by pro-inflammatory lymphocytes.

PubMed

Hodge, Greg; Roscioli, Eugene; Jersmann, Hubertus; Tran, Hai B; Holmes, Mark; Reynolds, Paul N; Hodge, Sandra

2016-10-21

Corticosteroid resistance is a major barrier to effective treatment of COPD. We have shown that the resistance is associated with decreased expression of glucocorticoid receptor (GCR) by senescent CD28nullCD8+ pro-inflammatory lymphocytes in peripheral blood of COPD patients. GCR must be bound to molecular chaperones heat shock proteins (Hsp) 70 and Hsp90 to acquire a high-affinity steroid binding conformation, and traffic to the nucleus. We hypothesized a loss of Hsp70/90 from these lymphocytes may further contribute to steroid resistance in COPD. Blood was collected from COPD (n = 10) and aged-matched controls (n = 10). To assess response to steroids, cytotoxic mediators, intracellular pro-inflammatory cytokines, CD28, GCR, Hsp70 and Hsp90 were determined in T and NKT-like cells in the presence of ± 10 μM prednisolone and 2.5 ng/mL cyclosporine A (binds to GCR-Hsp70/90 complex) using flow cytometry, western blot and fluorescence microscopy. A loss of expression of Hsp90 and GCR from CD28null CD8+ T and NKT-like cells in COPD was noted (Hsp70 unchanged). Loss of Hsp90 expression correlated with the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -0.763, p = 0.007 for T-cell IFNγ). Up-regulation of Hsp90 and associated decrease in pro-inflammatory cytokine production was found in CD28nullCD8+ T and NKT-like cells in the presence of 10 μM prednisolone and 2.5 ng/mL cyclosporine A. Loss of Hsp90 from cytotoxic/pro-inflammatory CD28nullCD8+ T and NKT-like cells could contribute to steroid resistance in COPD. Combination prednisolone and low-dose cyclosporine A therapy inhibits these pro-inflammatory cells and may reduce systemic inflammation in COPD.

Age-related changes in dorsal root ganglia, circulating and vascular calcitonin gene-related peptide (CGRP) concentrations in female rats: Effect of female sex steroid hormones

PubMed Central

Gangula, Pandu R.R.; Chauhan, Madhu; Reed, Luckey; Yallampalli, Chandra

2009-01-01

The aim of the present study is to investigate whether immunoreactive (I) calcitonin gene-related peptide (CGRP) content is decreased in plasma and mesenteric arteries (resistance arteries) in middle-aged rats and if so, whether sex steroid hormones enhance I-CGRP in middle-aged female rats. We also examined whether vascular CGRP receptor components, calcitonin receptor like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) are elevated by sex steroid hormones treatment in middle-aged female rats. Young adult (3 months old) and middle-aged (10–12 months old) ovariectomized rats were treated subcutaneously with estradiol-17β (E2; 2 mg), progesterone (P4; 5 mg), E2 +P4 (2 mg + 20 mg) or placebo (control). Radioimmunoassay and Western blot analysis were performed to measure I-CGRP content and CGRP receptor components in dorsal root ganglia (DRG), in resistance arteries and in plasma. Immunofluorescent staining methods were employed to determine cellular localization of CRLR, RAMP1 in resistance arteries. Our data demonstrated that I-CGRP content was significantly (p < 0.05) lower in the plasma and resistance arteries of middle-aged female rats compared to young controls. Both RAMP1 and CRLR were concentrated in vascular endothelium and the underlying smooth muscle cells. RAMP1 but not CRLR appeared to be decreased in middle-aged rat vasculature. Chronic perfusion of sex steroid hormones to ovariectomized rats: (1) significantly (p < 0.05) elevated I-CGRP in the DRG and in the plasma, and (2) significantly elevated RAMP1 (p < 0.05) but did not alter CRLR in resistance arteries. These data suggest that female sex steroid treatment enhances I-CGRP and its receptors, and thus regulate the blood pressure in aged female rats. PMID:19429067

Steroid hormone receptor status defines the MMTV promoter chromatin structure in vivo.

PubMed

Archer, T K; Fryer, C J; Lee, H L; Zaniewski, E; Liang, T; Mymryk, J S

1995-06-01

The ability to respond to small signalling molecules such as steroid hormones is important for many physiological processes. Steroid hormones act through a group of high affinity receptors that regulate transcription by binding to hormone response elements (HREs) located within the promoters of target genes, which themselves are organized with nuclear proteins to form chromatin. To dissect the mechanisms(s) of steroid hormone action we have used the steroid inducible mouse mammary tumor virus (MMTV) promoter as a model system. The MMTV promoter is assembled into a phased array of nucleosomes that are specifically positioned in rodent cells. Induction of transcription by glucocorticoids is accompanied by the appearance of a hypersensitive region in the proximal promoter which allows the hormone dependent assembly of a preinitiation complex including transcription factors such as nuclear factor 1 (NF1) and the octamer transcription factor (OTF). Surprisingly, when introduced by transient transfection, the progesterone receptor (PR) is unable to activate this promoter in vivo, a finding that may result from its inability to alter MMTV promoter chromatin. In an attempt to investigate the failure of the PR to activate the promoter, we have stably introduced the MMTV promoter into human T47D breast cancer cells that express high levels of the PR. In contrast to what has been observed previously in rodent cells, the MMTV templates resident in human breast cancer cells adopt a novel and constitutively open chromatin structure. The constitutively open chromatin structure is accompanied by the hormone independent loading of transcription factors including the PR and NF1. In T47D cells that stably express the glucocorticoid receptor, the MMTV promoter responds to glucocorticoids, but not progestins, and displays glucocorticoid induced restriction enzyme hypersensitivity and transcription factor loading. These findings suggest that the organization of the MMTV chromatin structure is dependent upon the cell type and receptor status of the recipient cell into which the MMTV promoter is stably introduced.

Cytokine modulation by glucocorticoids: mechanisms and actions in cellular studies.

PubMed

Brattsand, R; Linden, M

1996-01-01

Glucocorticoids inhibit the expression and action of most cytokines. This is part of the in vivo feed-back system between inflammation-derived cytokines and CNS-adrenal produced corticosteroids with the probable physiological relevance to balance parts of the host defence and anti-inflammatory systems of the body. Glucocorticoids modulate cytokine expression by a combination of genomic mechanisms. The activated glucocorticoid-receptor complex can (i) bind to and inactivate key proinflammatory transcription factors (e.g. AP-1, NF kappa B). This takes place at the promotor responsive elements of these factors, but has also been reported without the presence of DNA; (ii) via glucocorticoid responsive elements (GRE), upregulate the expression of cytokine inhibitory proteins, e.g. I kappa B, which inactivates the transcription factor NF kappa B and thereby the secondary expression of a series of cytokines; (iii) reduce the half-life time and utility of cytokine mRNAs. In studies with triggered human blood mononuclear cells in culture, glucocorticoids strongly diminish the production of the 'initial phase' cytokines IL-1 beta and TNF-alpha and the 'immunomodulatory' cytokines IL-2, IL-3, IL-4, IL-5, IL-10, IL-12 and IFN-gamma, as well as of IL-6, IL-8 and the growth factor GM-CSF. While steroid treatment broadly attenuates cytokine production, it cannot modulate it selectively, e.g. just the TH0, the TH1 or the TH2 pathways. The production of the 'anti-inflammatory' IL-10 is also inhibited. The exceptions of steroid down-regulatory activity on cytokine expression seem to affect 'repair phase' cytokines like TGF-beta and PDGF. These are even reported to be upregulated, which may explain the rather weak steroid dampening action on healing and fibrotic processes. Some growth factors, e.g. G-CSF and M-CSF, are only weakly affected. In addition to diminishing the production of a cytokine, steroids can also often inhibit its subsequent actions. Because cytokines work in cascades, this means that steroid treatment can block expression of the subsequent cytokines. The blocked cytokine activity does not depend on a reduced cytokine receptor expression; in fact available in vitro investigations show that while the cytokine expression is blunted, its receptor is upregulated. The cellular studies presented here may represent the maximum potential of steroids to modulate cytokine expression in human mononuclear cells. It remains to be determined by clinical-experimental studies how effective cytokine modulation can be achieved in situ in inflamed bowel by systemic or by topical steroid therapy. Such studies may also answer whether a blocked cytokine production/action is the key or just a secondary mechanism behind the unique efficacy of steroids in active inflammatory bowel disease.

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones

PubMed Central

Glass, Jennifer B.; Kretz, Cecilia B.; Ganesh, Sangita; Ranjan, Piyush; Seston, Sherry L.; Buck, Kristen N.; Landing, William M.; Morton, Peter L.; Moffett, James W.; Giovannoni, Stephen J.; Vergin, Kevin L.; Stewart, Frank J.

2015-01-01

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO3−, NO2−, Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu. PMID:26441925

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones.

PubMed

Glass, Jennifer B; Kretz, Cecilia B; Ganesh, Sangita; Ranjan, Piyush; Seston, Sherry L; Buck, Kristen N; Landing, William M; Morton, Peter L; Moffett, James W; Giovannoni, Stephen J; Vergin, Kevin L; Stewart, Frank J

2015-01-01

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO[Formula: see text], NO[Formula: see text], Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu.

Neuroactive steroids modulate HPA axis activity and cerebral brain-derived neurotrophic factor (BDNF) protein levels in adult male rats.

PubMed

Naert, Gaëlle; Maurice, Tangui; Tapia-Arancibia, Lucia; Givalois, Laurent

2007-01-01

Depression is characterized by hypothalamo-pituitary-adrenocortical (HPA) axis hyperactivity. In this major mood disorder, neurosteroids and neurotrophins, particularly brain-derived neurotrophic factor (BDNF), seem to be implicated and have some antidepressant effects. BDNF is highly involved in regulation of the HPA axis, whereas neurosteroids effects have never been clearly established. In this systematic in vivo study, we showed that the principal neuroactive steroids, namely dehydroepiandrosterone (DHEA), pregnenolone (PREG) and their sulfate esters (DHEA-S and PREG-S), along with allopregnanolone (ALLO), stimulated HPA axis activity, while also modulating central BDNF contents. In detail, DHEA, DHEA-S, PREG, PREG-S and ALLO induced corticotropin-releasing hormone (CRH) and/or arginine vasopressin (AVP) synthesis and release at the hypothalamic level, thus enhancing plasma adrenocorticotropin hormone (ACTH) and corticosterone (CORT) concentrations. This stimulation of the HPA axis occurred concomitantly with BDNF modifications at the hippocampus, amygdala and hypothalamus levels. We showed that these neurosteroids induced rapid effects, probably via neurotransmitter receptors and delayed effects perhaps after metabolization in other neuroactive steroids. We highlighted that they had peripheral effects directly at the adrenal level by inducing CORT release, certainly after estrogenic metabolization. In addition, we showed that, at the dose used, only DHEA, DHEA-S and PREG-S had antidepressant effects. In conclusion, these results highly suggest that part of the HPA axis and antidepressant effects of neuroactive steroids could be mediated by BDNF, particularly at the amygdala level. They also suggest that neurosteroids effects on central BDNF could partially explain the trophic properties of these molecules.

Tandem SAM Domain Structure of Human Caskin1: A Presynaptic, Self-Assembling Scaffold for CASK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stafford, Ryan L.; Hinde, Elizabeth; Knight, Mary Jane

2012-02-07

The synaptic scaffolding proteins CASK and Caskin1 are part of the fibrous mesh of proteins that organize the active zones of neural synapses. CASK binds to a region of Caskin1 called the CASK interaction domain (CID). Adjacent to the CID, Caskin1 contains two tandem sterile a motif (SAM) domains. Many SAM domains form polymers so they are good candidates for forming the fibrous structures seen in the active zone. We show here that the SAM domains of Caskin1 form a new type of SAM helical polymer. The Caskin1 polymer interface exhibits a remarkable segregation of charged residues, resulting in amore » high sensitivity to ionic strength in vitro. The Caskin1 polymers can be decorated with CASK proteins, illustrating how these proteins may work together to organize the cytomatrix in active zones.« less

Effects of the dietary amount and source of protein, resistance training and anabolic-androgenic steroids on body weight and lipid profile of rats.

PubMed

Aparicio, V A; Sánchez, C; Ortega, F B; Nebot, E; Kapravelou, G; Porres, J M; Aranda, P

2013-01-01

Dietary protein amount and source, hypertrophy resistance training (RT) and anabolicandrogenic steroids (AAS) may affect body weight and plasma and hepatic lipid profile. 157 adult male Wistar rats were randomly distributed in 16 experimental groups resulting in: normal-protein (NP) or high-protein (HP) diets, whey or soy-protein diets, with or without RT and with or without AAS, for 3 months. Final body weight was lower in the RT and AAS groups compared to sedentary and non- AAS groups, respectively (all, p<0.001). Plasma total cholesterol (TC) was lower for the HP compared to the NP diets, for the whey compared to the soy-protein diets and for the AAS compared to the non-AAS groups (all, p<0.001). Plasma HDL-cholesterol was higher in the RT groups (p<0.05) but lower for the AAS groups (p<0.001), the HP and the soy-protein diets (p<0.05). Plasma triglycerides (TAG) were lower for the HP diet (p<0.001), for the RT (p=0.002) and the non-AAS groups (p=0.001). Liver TC was lower for the NP (p<0.01), for the soyprotein (p<0.05) and for the AAS groups (p<0.001). Liver TAG were lower for the whey-protein diet (p<0.001), RT and non-AAS groups (both, p<0.05). Some interactions were found, such as the greater effect of AAS on reducing body weight of rats that performed RT or ingested a HP diet (all, p<0.05). HDL-cholesterol was higher when RT was combined with HP diets (p=0.010) or non-AAS and when HP diets were combined with non-AAS (both,p<0.001). Groups that combined RT with non-AAS administration obtained the lowest hepatic TAG (p<0.05). Among all the interventions tested, AAS was the factor that most negatively affected plasma and hepatic lipid profile, whereas HP diets and RT could benefit lipid profile, especially when combined. Copyright © AULA MEDICA EDICIONES 2013. Published by AULA MEDICA. All rights reserved.

6-Mercaptopurine-induced histopathological changes and xanthine oxidase expression in rat placenta.

PubMed

Taki, Kenji; Fukushima, Tamio; Ise, Ryota; Horii, Ikuo; Yoshida, Takemi

2012-01-01

The placenta secures the embryo and fetus to the endometrium and releases a variety of steroid and peptide hormones that convert the physiology of a female to that of a pregnant female. Chemical-induced alteration or deviation of placental function in the maternal and extraembryonic tissue can ultimately lead to pregnancy loss, congenital malformation and fetal death. The 6-mercaptopurine (6-MP), an anti-leukemic drug, is known to produce undesired effects on some organs, then the placenta/embryo toxicity of 6-MP was investigated in pregnant rats given 60 mg/kg with two intraperitoneal injections on gestation days (GD) 11 and 12. The rats were sacrificed and their placentas were collected on GD13 or 15. On GD15 small and limb-defected embryos were found in the 6-MP-treated rats. Placental weights were significantly reduced on GD15, as well as a reduced number of cells was detected in the labyrinth zone with both the labyrinth and basal zones having thinned. Cleaved caspase-3-positive cells increased in number in the labyrinth zone, while in the basal zone, glycogen cells reduced with cytolysis. The number of spongiotrophoblasts and trophoblastic giant cells also increased by 6-MP treatment. The 6-MP-treatment resulted in the increased xanthine oxidase (Xdh) expression in the placenta, which gene is related to the ischemic condition of tissues. These data suggest that apoptosis of the labyrinth zone cells may lead to decreased materno-fetal exchange. Moreover, subsequent ischemia in the placental tissue may occur and induce Xdh expression.

Functional Zonation of the Adult Mammalian Adrenal Cortex

PubMed Central

Vinson, Gavin P.

2016-01-01

The standard model of adrenocortical zonation holds that the three main zones, glomerulosa, fasciculata, and reticularis each have a distinct function, producing mineralocorticoids (in fact just aldosterone), glucocorticoids, and androgens respectively. Moreover, each zone has its specific mechanism of regulation, though ACTH has actions throughout. Finally, the cells of the cortex originate from a stem cell population in the outer cortex or capsule, and migrate centripetally, changing their phenotype as they progress through the zones. Recent progress in understanding the development of the gland and the distribution of steroidogenic enzymes, trophic hormone receptors, and other factors suggests that this model needs refinement. Firstly, proliferation can take place throughout the gland, and although the stem cells are certainly located in the periphery, zonal replenishment can take place within zones. Perhaps more importantly, neither the distribution of enzymes nor receptors suggest that the individual zones are necessarily autonomous in their production of steroid. This is particularly true of the glomerulosa, which does not seem to have the full suite of enzymes required for aldosterone biosynthesis. Nor, in the rat anyway, does it express MC2R to account for the response of aldosterone to ACTH. It is known that in development, recruitment of stem cells is stimulated by signals from within the glomerulosa. Furthermore, throughout the cortex local regulatory factors, including cytokines, catecholamines and the tissue renin-angiotensin system, modify and refine the effects of the systemic trophic factors. In these and other ways it more and more appears that the functions of the gland should be viewed as an integrated whole, greater than the sum of its component parts. PMID:27378832

The Steroid Hormone 20-Hydroxyecdysone Regulates the Conjugation of Autophagy-Related Proteins 12 and 5 in a Concentration and Time-Dependent Manner to Promote Insect Midgut Programmed Cell Death

PubMed Central

Li, Yong-Bo; Yang, Ting; Wang, Jin-Xing; Zhao, Xiao-Fan

2018-01-01

Autophagy requires the conjugation of autophagy-related protein 12 (ATG12) to autophagy-related protein 5 (ATG5) through covalent attachment. However, the signals regulating ATG12–ATG5 conjugation are unclear. The larval midgut of lepidopteran insects performs autophagy and apoptosis sequentially during the transition of larvae to pupae under regulation by the steroid hormone 20-hydroxyecdysone (20E), thus representing a model to study steroid hormone regulation of ATG12–ATG5 conjugation. In the present study, using the lepidopteran insect Helicoverpa armigera as a model, we report that 20E regulates the conjugation of ATG12–ATG5 in a concentration and time-dependent manner. The ATG12–ATG5 conjugate was abundant in the epidermis, midgut, and fat body during metamorphosis from the larvae to the pupae; however, the ATG12–ATG5 conjugate level decreased at the time of pupation. At low concentrations (2–5 µM) over a short time course (1–48 h), 20E promoted the conjugation of ATG12–ATG5; however, at 10 µM and 72 h, 20E repressed the conjugation of ATG12–ATG5. ATG12 was localized in the larval midgut during metamorphosis. Knockdown of ATG12 in larvae caused death with delayed pupation, postponed the process of midgut programmed cell death (PCD), and repressed ATG8 (also called LC3-I) transformation to LC3-II and the cleavage of caspase-3; therefore, knockdown of ATG12 in larvae blocked both autophagy and apoptosis. Knockdown of ATG12 in H. armigera epidermis cell line cells also repressed 20E-induced autophagosome formation and caspase-3 activation. The results suggested that 20E plays key role in the regulation of ATG12–ATG5 conjugation in a concentration and time-dependent manner for autophagy or apoptosis, and that ATG12 is necessary by both autophagy and apoptosis during insect midgut PCD. PMID:29467720

Identification and functional analysis of cytochrome P450 complement in Streptomyces virginiae IBL14

PubMed Central

2013-01-01

Background As well known, both natural and synthetic steroidal compounds are powerful endocrine disrupting compounds (EDCs) which can cause reproductive toxicity and affect cellular development in mammals and thus are generally regarded as serious contributors to water pollution. Streptomyces virginiae IBL14 is an effective degradative strain for many steroidal compounds and can also catalyze the C25 hydroxylation of diosgenin, the first-ever biotransformation found on the F-ring of diosgenin. Results To completely elucidate the hydroxylation function of cytochrome P450 genes (CYPs) found during biotransformation of steroids by S. virginiae IBL14, the whole genome sequencing of this strain was carried out via 454 Sequencing Systems. The analytical results of BLASTP showed that the strain IBL14 contains 33 CYPs, 7 ferredoxins and 3 ferredoxin reductases in its 8.0 Mb linear chromosome. CYPs from S. virginiae IBL14 are phylogenetically closed to those of Streptomyces sp. Mg1 and Streptomyces sp. C. One new subfamily was found as per the fact that the CYP Svu001 in S. virginiae IBL14 shares 66% identity only to that (ZP_05001937, protein identifer) from Streptomyces sp. Mg1. Further analysis showed that among all of the 33 CYPs in S. virginiae IBL14, three CYPs are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three CYPs with ATP/GTP binding proteins, four CYPs arranged with transcriptional regulatory genes and one CYP located on the upstream of an ATP-binding protein and transcriptional regulators as well as four CYPs associated with other functional genes involved in secondary metabolism and degradation. Conclusions These characteristics found in CYPs from S. virginiae IBL14 show that the EXXR motif in the K-helix is not absolutely conserved in CYP157 family and I-helix not absolutely essential for the CYP structure, too. Experimental results showed that both CYP Svh01 and CYP Svu022 are two hydroxylases, capable of bioconverting diosgenone into isonuatigenone and β-estradiol into estriol, respectively. PMID:23442312

Reference ranges for urinary concentrations and ratios of endogenous steroids, which can be used as markers for steroid misuse, in a Caucasian population of athletes.

PubMed

Van Renterghem, Pieter; Van Eenoo, Peter; Geyer, Hans; Schänzer, Wilhelm; Delbeke, Frans T

2010-02-01

The detection of misuse with naturally occurring steroids is a great challenge for doping control laboratories. Intake of natural anabolic steroids alters the steroid profile. Thus, screening for exogenous use of these steroids can be established by monitoring a range of endogenous steroids, which constitute the steroid profile, and evaluate their concentrations and ratios against reference ranges. Elevated values of the steroid profile constitute an atypical finding after which a confirmatory IRMS procedure is needed to unequivocally establish the exogenous origin of a natural steroid. However, the large inter-individual differences in urinary steroid concentrations and the recent availability of a whole range of natural steroids (e.g. dehydroepiandrosterone and androstenedione) which each exert a different effect on the monitored parameters in doping control complicate the interpretation of the current steroid profile. The screening of an extended steroid profile can provide additional parameters to support the atypical findings and can give specific information upon the steroids which have been administered. The natural concentrations of 29 endogenous steroids and 11 ratios in a predominantly Caucasian population of athletes were determined. The upper reference values at 97.5%, 99% and 99.9% levels were assessed for male (n=2027) and female (n=1004) populations. Monitoring minor metabolites and evaluation of concentration ratios with respect to their natural abundances could improve the interpretation of the steroid profile in doping analysis. Copyright 2009 Elsevier Inc. All rights reserved.

Serum proteins by capillary zone electrophoresis: approaches to the definition of reference values.

PubMed

Petrini, C; Alessio, M G; Scapellato, L; Brambilla, S; Franzini, C

1999-10-01

The Paragon CZE 2000 (Beckman Analytical, Milan, Italy) is an automatic dedicated capillary zone electrophoresis (CZE) system, producing a five-zone serum protein pattern with quantitative estimation of the zones. With the view of substituting this instrument for two previously used serum protein electrophoresis techniques, we planned to produce reference values for the "new" systems leading to compatible interpretation of the results. High resolution cellulose acetate electrophoresis with visual inspection and descriptive reporting (HR-CAE) and five-zone cellulose acetate electrophoresis with densitometry (CAE-D) were the previously used techniques. Serum samples (n = 167) giving "normal pattern" with HR-CAE were assayed with the CZE system, and the results were statistically assessed to yield 0.95 reference intervals. One thousand normal and pathological serum samples were then assayed with the CAE-D and the CZE techniques, and the regression equations of the CAE-D values over the CZE values for the five zones were used to transform the CAE-D reference limits into the CZE reference limits. The two sets of reference values thereby produced were in good agreement with each other and also with reference values previously reported for the CZE system. Thus, reference values for the CZE techniques permit interpretation of results coherent with the previously used techniques and reporting modes.

Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka

The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6more » but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.« less

Transformation of glucocorticoid receptors bound to the antagonist RU 486: Effects of alkaline phosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruol, D.J.; Wolfe, K.A.

1990-08-28

RU 486 is a synthetic steroid that binds avidly to glucocorticoid receptors without promoting their transformation into activated transcription factors. A significant part of this behavior has been shown to be due to a failure of the RU 486 bound receptor to be efficiently released from a larger (sedimenting at 8-9 S) multimeric complex containing the 90-kDa heat shock protein. The studies have found that in vitro at 15{degree}C the RU 486-receptor was slowly released from the 8-9S complex and converted into a DNA binding protein by a process that could be blocked by sodium fluoride. Moreover, this transition wasmore » significantly accelerated by treatment with alkaline phosphatase. High-resolution anion-exchange chromatography showed that the profile of receptor subspecies released from the 8-9S complex was different for the RU 486 bound receptor when compared to the receptor occupied by the agonist triamcinolone acetonide. Production of the earliest eluting receptor form (peak A) was inhibited with RU 486. Treatment of the Ru 486-receptor with alkaline phosphatase increased the formation of the peak A subspecies as well as the capacity of receptor to bind DNA-cellulose. Taken together, the results indicate that phosphorylation of the receptor or a tightly bound factor contributes to defining the capacity with which individual steroids can promote dissociation of the 8-9S complex and conversion of the glucocorticoid receptor into a DNA-binding protein.« less

Predictors of natively unfolded proteins: unanimous consensus score to detect a twilight zone between order and disorder in generic datasets.

PubMed

Deiana, Antonio; Giansanti, Andrea

2010-04-21

Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and applicative studies. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded ones. In this work we propose an operational method to identify proteins belonging to the twilight zone by combining into a consensus score good performing single predictors of folding. In this methodological paper dichotomic folding indexes are considered: hydrophobicity-charge, mean packing, mean pairwise energy, Poodle-W and a new global index, that is called here gVSL2, based on the local disorder predictor VSL2. The performance of these indexes is evaluated on different datasets, in particular on a new dataset composed by 2369 folded and 81 natively unfolded proteins. Poodle-W, gVSL2 and mean pairwise energy have good performance and stability in all the datasets considered and are combined into a strictly unanimous combination score SSU, that leaves proteins unclassified when the consensus of all combined indexes is not reached. The unclassified proteins: i) belong to an overlap region in the vector space of amino acidic compositions occupied by both folded and unfolded proteins; ii) are composed by approximately the same number of order-promoting and disorder-promoting amino acids; iii) have a mean flexibility intermediate between that of folded and that of unfolded proteins. Our results show that proteins unclassified by SSU belong to a twilight zone. Proteins left unclassified by the consensus score SSU have physical properties intermediate between those of folded and those of natively unfolded proteins and their structural properties and evolutionary history are worth to be investigated.

Predictors of natively unfolded proteins: unanimous consensus score to detect a twilight zone between order and disorder in generic datasets

PubMed Central

2010-01-01

Background Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and applicative studies. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded ones. In this work we propose an operational method to identify proteins belonging to the twilight zone by combining into a consensus score good performing single predictors of folding. Results In this methodological paper dichotomic folding indexes are considered: hydrophobicity-charge, mean packing, mean pairwise energy, Poodle-W and a new global index, that is called here gVSL2, based on the local disorder predictor VSL2. The performance of these indexes is evaluated on different datasets, in particular on a new dataset composed by 2369 folded and 81 natively unfolded proteins. Poodle-W, gVSL2 and mean pairwise energy have good performance and stability in all the datasets considered and are combined into a strictly unanimous combination score SSU, that leaves proteins unclassified when the consensus of all combined indexes is not reached. The unclassified proteins: i) belong to an overlap region in the vector space of amino acidic compositions occupied by both folded and unfolded proteins; ii) are composed by approximately the same number of order-promoting and disorder-promoting amino acids; iii) have a mean flexibility intermediate between that of folded and that of unfolded proteins. Conclusions Our results show that proteins unclassified by SSU belong to a twilight zone. Proteins left unclassified by the consensus score SSU have physical properties intermediate between those of folded and those of natively unfolded proteins and their structural properties and evolutionary history are worth to be investigated. PMID:20409339

ent-Steroids: novel tools for studies of signaling pathways.

PubMed

Covey, Douglas F

2009-07-01

Membrane receptors are often modulated by steroids and it is necessary to distinguish the effects of steroids at these receptors from effects occurring at nuclear receptors. Additionally, it may also be mechanistically important to distinguish between direct effects caused by binding of steroids to membrane receptors and indirect effects on membrane receptor function caused by steroid perturbation of the membrane containing the receptor. In this regard, ent-steroids, the mirror images of naturally occurring steroids, are novel tools for distinguishing between these various actions of steroids. The review provides a background for understanding the different actions that can be expected of steroids and ent-steroids in biological systems, references for the preparation of ent-steroids, a short discussion about relevant forms of stereoisomerism and the requirements that need to be fulfilled for the interaction between two molecules to be enantioselective. The review then summarizes results of biophysical, biochemical and pharmacological studies published since 1992 in which ent-steroids have been used to investigate the actions of steroids in membranes and/or receptor-mediated signaling pathways.

ent-Steroids: Novel Tools for Studies of Signaling Pathways

PubMed Central

Covey, Douglas F.

2008-01-01

Membrane receptors are often modulated by steroids and it is necessary to distinguish the effects of steroids at these receptors from effects occurring at nuclear receptors. Additionally, it may also be mechanistically important to distinguish between direct effects caused by binding of steroids to membrane receptors and indirect effects on membrane receptor function caused by steroid perturbation of the membrane containing the receptor. In this regard, ent-steroids, the mirror images of naturally occurring steroids, are novel tools for distinguishing between these various actions of steroids. The review provides a background for understanding the different actions that can be expected of steroids and ent-steroids in biological systems, references for the preparation of ent-steroids, a short discussion about relevant forms of stereoisomerism and the requirements that need to be fulfilled for the interaction between two molecules to be enantioselective. The review then summarizes results of biophysical, biochemical and pharmacological studies published since 1992 in which ent-steroids have been used to investigate the actions of steroids in membranes and/or receptor-mediated signaling pathways. PMID:19103212

Clinical applications of MS-based protein quantification.

PubMed

Sabbagh, Bassel; Mindt, Sonani; Neumaier, Michael; Findeisen, Peter

2016-04-01

Mass spectrometry-based assays are increasingly important in clinical laboratory medicine and nowadays are already commonly used in several areas of routine diagnostics. These include therapeutic drug monitoring, toxicology, endocrinology, pediatrics, and microbiology. Accordingly, some of the most common analyses are therapeutic drug monitoring of immunosuppressants, vitamin D, steroids, newborn screening, and bacterial identification. However, MS-based quantification of peptides and proteins for routine diagnostic use is rather rare up to now despite excellent analytical specificity and good sensitivity. Here, we want to give an overview over current fit-for-purpose assays for MS-based protein quantification. Advantages as well as challenges of this approach will be discussed with focus on feasibility for routine diagnostic use. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Final Report: Proteomic study of brassinosteroid responses in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhiyong; Burlingame, Alma

2017-11-29

The steroid hormone brassinosteroid (BR) is a major growth-promoting phytohormone. The specific aim of the current project is to identify BR-regulated proteins and characterize their functions in various aspects of plant growth, development, and adaptation. Our research has significantly advanced our understanding of how BR signal is transduced from the receptor at the cell surface to changes of nuclear gene expression and other cellular responses such as vesicle trafficking, as well as developmental transitions such as seed germination and flowering. We have also developed effective proteomic methods for quantitative analysis of protein phosphorylation and for identification of glycosylated proteins. Throughmore » this DOE funding, we have performed several proteomic experiments and made major discoveries.« less

Impact of Intraoperative Steroids on Postoperative Infection Rates and Length of Hospital Stay: A Study of 1200 Spine Surgery Patients.

PubMed

Elsamadicy, Aladine A; Wang, Timothy Y; Back, Adam G; Sergesketter, Amanda; Warwick, Hunter; Karikari, Isaac O; Gottfried, Oren N

2016-12-01

The use of intraoperative steroids and their effects are relatively unknown and remain controversial. The aim of this study was to determine the effects of intraoperative steroid use on postoperative complications and length of hospital stay after spine surgery. Medical records of 1200 adult patients undergoing spine surgery at Duke University Medical Center during the period 2008-2010 were retrospectively reviewed; 495 (41.25%) patients were administered intraoperative steroids, and 705 (58.75%) patients were not administered intraoperative steroids. Patient demographics, comorbidities, and postoperative complication rates were collected. The primary outcomes investigated were postoperative complications, specifically length of hospital stay and infection rates. Patient demographics were similar between both cohorts. Comorbidities were also similar, with the intraoperative steroid use cohort having a higher number of patients with long-term steroid use than the no intraoperative steroid use cohort (6.95% [no steroids] vs. 13.74% [steroid use], P < 0.001). Operative variables, including length of operation and median number of fusion levels operated, were also similar between the 2 groups. Lumbar spine was the most common surgical location. Patients who were administered intraoperative steroids had a shorter length of hospital stay by an average of 1 day (6.06 days ± 6.76 [no steroids] vs. 5.04 days ± 4.86 [steroid use], P = 0.0025), lower rates of urinary tract infections (10.37% [no steroids] vs. 6.88% [steroid use], P = 0.040), and lower rates of other infections that were not deep or superficial surgical site infections (9.22% [no steroids] vs. 6.06% [steroid use], P = 0.0460). Patients who receive intraoperative steroids have shorter hospital stays and lower infection rates after spine surgery. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel mechanisms for DHEA action.

PubMed

Prough, Russell A; Clark, Barbara J; Klinge, Carolyn M

2016-04-01

Dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA), secreted by the adrenal cortex, gastrointestinal tract, gonads, and brain, and its sulfated metabolite DHEA-S are the most abundant endogeneous circulating steroid hormones. DHEA actions are classically associated with age-related changes in cardiovascular tissues, female fertility, metabolism, and neuronal/CNS functions. Early work on DHEA action focused on the metabolism to more potent sex hormones, testosterone and estradiol, and the subsequent effect on the activation of the androgen and estrogen steroid receptors. However, it is now clear that DHEA and DHEA-S act directly as ligands for many hepatic nuclear receptors and G-protein-coupled receptors. In addition, it can function to mediate acute cell signaling pathways. This review summarizes the molecular mechanisms by which DHEA acts in cells and animal models with a focus on the 'novel' and physiological modes of DHEA action. © 2016 Society for Endocrinology.

Analgesic and antipyretic activities of Momordica charantia Linn. fruits

PubMed Central

Patel, Roshan; Mahobia, Naveen; Upwar, Nitin; Waseem, Naheed; Talaviya, Hetal; Patel, Zalak

2010-01-01

Plant Momordica charantia Linn. belongs to family Cucurbitaceae. It is known as bitter gourd in English and karela in Hindi. Earlier claims show that the plant is used in stomachic ailments as a carminative tonic; as an antipyretic and antidiabetic agent; and in rheumatoid arthritis and gout. The fruit has been claimed to contain charantin, steroidal saponin, momordium, carbohydrates, mineral matters, ascorbic acid, alkaloids, glucosides, etc. The ethanolic extract of the fruit showed the presence of alkaloids, tannins, glycosides, steroids, proteins, and carbohydrates. The present study was carried out using acetic acid-induced writhing and tail-immersion tests in mice, while yeast-induced pyrexia in rats. The ethanolic extracts (250 and 500 mg/kg, po.) showed an analgesic and antipyretic effect, which was significantly higher than that in the control rats. The observed pharmacological activities provide the scientific basis to support traditional claims as well as explore some new and promising leads. PMID:22247882

Analgesic and antipyretic activities of Momordica charantia Linn. fruits.

PubMed

Patel, Roshan; Mahobia, Naveen; Upwar, Nitin; Waseem, Naheed; Talaviya, Hetal; Patel, Zalak

2010-10-01

Plant Momordica charantia Linn. belongs to family Cucurbitaceae. It is known as bitter gourd in English and karela in Hindi. Earlier claims show that the plant is used in stomachic ailments as a carminative tonic; as an antipyretic and antidiabetic agent; and in rheumatoid arthritis and gout. The fruit has been claimed to contain charantin, steroidal saponin, momordium, carbohydrates, mineral matters, ascorbic acid, alkaloids, glucosides, etc. The ethanolic extract of the fruit showed the presence of alkaloids, tannins, glycosides, steroids, proteins, and carbohydrates. The present study was carried out using acetic acid-induced writhing and tail-immersion tests in mice, while yeast-induced pyrexia in rats. The ethanolic extracts (250 and 500 mg/kg, po.) showed an analgesic and antipyretic effect, which was significantly higher than that in the control rats. The observed pharmacological activities provide the scientific basis to support traditional claims as well as explore some new and promising leads.

Cytotoxic and apoptosis-inducing activity of C21 steroids from the roots of Cynanchum atratum.

PubMed

Zhang, Jian; Ma, Lin; Wu, Zheng-Feng; Yu, Shu-Le; Wang, Lei; Ye, Wen-Cai; Zhang, Qing-Wen; Yin, Zhi-Qi

2017-06-01

Two new (1-2) and two known C 21 steroids (3-4) were isolated from the roots of Cynanchum atratum. Their structures were elucidated by detailed 1D and 2D spectroscopic. The MTT assay showed that compounds 1-4 displayed obvious cytotoxic activities against HepG2 cells with IC 50 values ranging from 10.19μM to 76.12μM. Compounds 1-3 also exhibited cytotoxic effects in A549 cells with IC 50 values of 30.87-95.39μM. Compound 3 showed the antiproliferative activity via G0/G1 cell cycle arrest and proapoptosis in HepG2 cells by Flowcytometry analysis. Western blotting analysis revealed that compound 3 could induce HepG2 cell apoptosis via the mitochondrial pathway by downregulating Bcl-2 expression, upregulating Bax protein expression, and activating caspase-9 and caspase-3. Copyright © 2017 Elsevier Inc. All rights reserved.

Progesterone Modulates a Neuronal Nicotinic Acetylcholine Receptor

NASA Astrophysics Data System (ADS)

Valera, S.; Ballivet, M.; Bertrand, D.

1992-10-01

The major brain nicotinic acetylcholine receptor is assembled from two subunits termed α 4 and nα 1. When expressed in Xenopus oocytes, these subunits reconstitute a functional acetylcholine receptor that is inhibited by progesterone levels similar to those found in serum. In this report, we show that the steroid interacts with a site located on the extracellular part of the protein, thus confirming that inhibition by progesterone is not due to a nonspecific perturbation of the membrane bilayer or to the activation of second messengers. Because inhibition by progesterone does not require the presence of agonist, is voltage-independent, and does not alter receptor desensitization, we conclude that the steroid is not an open channel blocker. In addition, we show that progesterone is not a competitive inhibitor but may interact with the acetylcholine binding site and that its effect is independent of the ionic permeability of the receptor.

[Sub-acute encephalopathy that responds to steroids without any evidence of autoimmune thyroid disease: a case of non-vasculitic autoimmune meningoencephalitis].

PubMed

López-Ariztegui, N; Lobato-Casado, P; Muñoz-Escudero, F; Polo-Martín, M; Montes-Gonzalo, M C; Alvarez-Tejerina, A

To report a case of sub-acute encephalopathy with all the extension study negative and with response to steroid therapy. The study involves a 22-year-old female with no relevant past history who presented symptoms of sub-acute encephalopathy consisting in behavioural disorders, generalised seizures and bradypsychia, which gradually progressed to a state of low-level consciousness. While she was in hospital all kinds of diagnostic tests were conducted, the results of which were either normal or negative; the electroencephalogram was repeatedly abnormal and detection of protein 14-3-3 in cerebrospinal fluid was positive. Empirical corticoid therapy was begun with clinical and electrophysiological improvements and the patient recovered completely without any sequelae. With no evidence of autoimmune thyroid disease, although non-specific autoimmunity was present, the patient was diagnosed as having non-vasculitic autoimmune meningoencephalitis.

Therapeutic Potential for Targeting the Suppressor of Cytokine Signaling-1 pathway for the treatment of SLE

PubMed Central

Sukka-Ganesh, Bhagyalaxmi; Larkin, Joseph

2016-01-01

Although the specific events dictating systemic lupus erythematosus (SLE) pathology remain unclear, abundant evidence indicates a critical role for dysregulated cytokine signaling in disease progression. Notably, the suppressor of cytokine signaling (SOCS) family of intracellular proteins, in particular the kinase inhibitory region (KIR) bearing SOCS1 and SOCS3, play a critical role in regulating cytokine signaling. To assess a relationship between SOCS1/SOCS3 expression and SLE, the goals of this study were to: 1) evaluate the time kinetics of SOCS1/SOCS3 message and protein expression based on SLE associated stimulations, 2) compare levels of SOCS1 and SOCS3 present in SLE patients and healthy controls by message and protein, 3) relate SOCS1/SOCS3 expression to inflammatory markers in SLE patients, and 4) correlate SOCS1/SOCS3 levels to current treatments. We found that SOCS1 and SOCS3 were most abundant in murine splenic samples at 48 hours subsequent to stimulation by anti-CD3, LPS, or interferon gamma. In addition, significant reductions in SOCS1 and SOCS3 were present within PMBC’s of SLE patients compared to controls by both mRNA and protein expression. We also found that decreased levels of SOCS1 in SLE patients were correlated to enhanced levels of inflammatory markers and up-regulated expression of MHC class II. Finally, we show that patients receiving steroid treatment possessed higher levels SOCS1 compared to SLE patient counterparts, and that steroid administration to human PBMCs up-regulated SOCS1 message in a dose and time dependent manner. Together, these results suggest that therapeutic strategies focused on SOCS1 signaling may have efficacy in the treatment of SLE. PMID:27781323

Hydrocortisone activation of human herpesvirus 8 viral DNA replication and gene expression in vitro.

PubMed

Hudnall, S D; Rady, P L; Tyring, S K; Fish, J C

1999-03-15

Patients undergoing chronic steroid therapy for organ transplantation are at increased risk for development of human herpes virus 8(HHV-8)-associated Kaposi's sarcoma (KS). It has also been reported that following steroid withdrawal, KS lesions often undergo partial or complete regression. We have examined the effect of corticosteroid treatment on HHV-8 replication, gene expression, and lytic protein expression in BCBL-1 cells in vitro. BCBL-1 cells were collected after culture for 24-72 hr with hydrocortisone (HC) 1-5 microM, phorbol ester 20 ng/ml (positive control), and culture medium only (negative control). HHV-8 genomic conformation was examined by Gardella gel analysis. mRNA expression of viral cyclin (v-Cyc), viral Bcl-2 (v-Bcl-2), viral macrophage inflammatory protein-I (v-MIP-I), viral interferon regulatory factor-1(v-IRF-1), and viral tegument protein (TP) was examined by RT-PCR Southern blot. Viral protein expression within the cells was examined by indirect immunofluorescence using 5 different HHV-8 positive antisera from 4 renal transplant recipients and 1 patient with classic KS. Gardella gel analysis revealed that HC induced an accumulation of the linear replicative genomic form of the virus in a time-dependent fashion. Southern blot analysis of the RT-PCR products revealed that HC induced increased expression of v-IRF-1, v-Bcl-2, and TP mRNA, with little discernible effect on v-Cyc, and v-MIP-I. Immunofluorescence revealed that HC induced increased numbers of cells expressing lytic antigens. These data indicate that hydrocortisone acts directly on BCBL-1 cells to activate the lytic cycle of HHV-8 and provide further support for the hypothesis that HHV-8 is activated in corticosteroid-treated immunocompromised patients.

The pure estrogen receptor antagonist ICI 182,780 promotes a novel interaction of estrogen receptor-alpha with the 3',5'-cyclic adenosine monophosphate response element-binding protein-binding protein/p300 coactivators.

PubMed

Jaber, Basem M; Gao, Tong; Huang, Luping; Karmakar, Sudipan; Smith, Carolyn L

2006-11-01

Estrogen receptor-alpha (ERalpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Abundant evidence demonstrates that ERalpha agonists promote, whereas antagonists inhibit, receptor binding to coactivators. In this report we demonstrate that binding of the ICI 182,780 (ICI) pure antiestrogen to ERalpha promotes its interaction with the cAMP response element-binding protein-binding protein (CBP)/p300 but not the p160 family of coactivators, demonstrating the specificity of this interaction. Amino acid mutations within the coactivator binding surface of the ERalpha ligand-binding domain revealed that CBP binds to this region of the ICI-liganded receptor. The carboxy-terminal cysteine-histidine rich domain 3 of CBP, rather than its amino-terminal nuclear interacting domain, shown previously to mediate agonist-dependent interactions of CBP with nuclear receptors, is required for binding to ICI-liganded ERalpha. Chromatin immunoprecipitation assays revealed that ICI but not the partial agonist/antagonist 4-hydroxytamoxifen is able to recruit CBP to the pS2 promoter, and this distinguishes ICI from this class of antiestrogens. Chromatin immunoprecipitation assays for pS2 and cytochrome P450 1B1 promoter regions revealed that ICI-dependent recruitment of CBP, but not receptor, to ERalpha targets is gene specific. ICI treatment did not recruit the steroid receptor coactivator 1 to the pS2 promoter, and it failed to induce the expression of this gene. Taken together, these data indicate that recruitment of the CBP coactivator/cointegrator without steroid receptor coactivator 1 to ERalpha is insufficient to promote transcription of ERalpha target genes.

Overexpression of a novel MADS-box gene SlFYFL delays senescence, fruit ripening and abscission in tomato

PubMed Central

Xie, Qiaoli; Hu, Zongli; Zhu, Zhiguo; Dong, Tingting; Zhao, Zhiping; Cui, Baolu; Chen, Guoping

2014-01-01

MADS-domain proteins are important transcription factors involved in many biological processes of plants. In our study, a tomato MADS-box gene, SlFYFL, was isolated. SlFYFL is expressed in all tissues of tomato and significantly higher in mature leave, fruit of different stages, AZ (abscission zone) and sepal. Delayed leaf senescence and fruit ripening, increased storability and longer sepals were observed in 35S:FYFL tomato. The accumulation of carotenoid was reduced, and ethylene content, ethylene biosynthetic and responsive genes were down-regulated in 35S:FYFL fruits. Abscission zone (AZ) did not form normally and abscission zone development related genes were declined in AZs of 35S:FYFL plants. Yeast two-hybrid assay revealed that SlFYFL protein could interact with SlMADS-RIN, SlMADS1 and SlJOINTLESS, respectively. These results suggest that overexpression of SlFYFL regulate fruit ripening and development of AZ via interactions with the ripening and abscission zone-related MADS box proteins. PMID:24621662

BAR Proteins PSTPIP1/2 Regulate Podosome Dynamics and the Resorption Activity of Osteoclasts

PubMed Central

Sztacho, Martin; Segeletz, Sandra; Sanchez-Fernandez, Maria Arantzazu; Czupalla, Cornelia; Niehage, Christian; Hoflack, Bernard

2016-01-01

Bone resorption in vertebrates relies on the ability of osteoclasts to assemble F-actin-rich podosomes that condense into podosomal belts, forming sealing zones. Sealing zones segregate bone-facing ruffled membranes from other membrane domains, and disassemble when osteoclasts migrate to new areas. How podosome/sealing zone dynamics is regulated remains unknown. We illustrate the essential role of the membrane scaffolding F-BAR-Proline-Serine-Threonine Phosphatase Interacting Proteins (PSTPIP) 1 and 2 in this process. Whereas PSTPIP2 regulates podosome assembly, PSTPIP1 regulates their disassembly. PSTPIP1 recruits, through its F-BAR domain, the protein tyrosine phosphatase non-receptor type 6 (PTPN6) that de-phosphophorylates the phosphatidylinositol 5-phosphatases SHIP1/2 bound to the SH3 domain of PSTPIP1. Depletion of any component of this complex prevents sealing zone disassembly and increases osteoclast activity. Thus, our results illustrate the importance of BAR domain proteins in podosome structure and dynamics, and identify a new PSTPIP1/PTPN6/SHIP1/2-dependent negative feedback mechanism that counterbalances Src and PI(3,4,5)P3 signalling to control osteoclast cell polarity and activity during bone resorption. PMID:27760174

[Signal transduction mechanisms of hormones through membrane receptors].

PubMed

Yasufuku-Takano, Junko; Takano, Koji

2002-02-01

Hormones exert their effect on cells either via membrane receptors or intracellular receptors. This paper aims to review membrane receptors and the intracellular signal transduction mechanisms. Membrane receptors could be classified according to their structural characteristics and the way they initiate the intracellular signal transduction. These include 1) Seven transmembrane(or G-protein coupled) receptors--heterotrimeric G-proteins--effector, system, 2) Receptor tyrosine kinases--protein-protein interaction through SH2, SH3, and PTB domain--MAP kinase cascades and PI3-kinase pathways, 3) Cytokine receptors--JAK--STAT pathways, 4) Receptors of the TGF- beta superfamily--SMAD pathways, 5) Apoptosis-related receptors--caspase pathways, and 6) ligand-gated ion channels. There are growing knowledge of cross-talks between these pathways. It is being recognized that steroid hormones have distinct membrane receptors, which mediate rapid, nongenomic effect.

Interactions of trans-acting factor(s) with the estradiol response element and nuclear factor 1 of the vitellogenin II gene of Japanese quail.

PubMed

Gupta, S; Upadhayay, R; Kanungo, M S

1996-08-01

This study was directed at achieving an understanding of the mechanisms by which steroid hormones control the synthesis of vitellogenin (VTG) protein in the liver of the Japanese quail. Northern hybridization shows that administration of estradiol alone or with progesterone stimulates the synthesis of VTG mRNA. Gel mobility shift assay of DNA fragments containing the ERE and NF 1 shows that estradiol alone or with progesterone increases the levels of nuclear proteins that bind to these cis-acting elements of the promoter of the VTG gene. The cooperative effect of the two hormones seen at the level of expression of the VTG gene may be due to protein-protein interactions of trans-acting factors that bind to ERE and NF 1.

Downregulation of peroxisome proliferator-activated receptors (PPARs) in nasal polyposis.

PubMed

Cardell, Lars-Olaf; Hägge, Magnus; Uddman, Rolf; Adner, Mikael

2005-11-07

Peroxisome proliferator-activated receptor (PPAR) alpha, betadelta and gamma are nuclear receptors activated by fatty acid metabolites. An anti-inflammatory role for these receptors in airway inflammation has been suggested. Nasal biopsies were obtained from 10 healthy volunteers and 10 patients with symptomatic allergic rhinitis. Nasal polyps were obtained from 22 patients, before and after 4 weeks of local steroid treatment (fluticasone). Real-time RT-PCR was used for mRNA quantification and immunohistochemistry for protein localization and quantification. mRNA expression of PPARalpha, PPARbetadelta, PPARgamma was found in all specimens. No differences in the expression of PPARs were obtained in nasal biopsies from patients with allergic rhinitis and healthy volunteers. Nasal polyps exhibited lower levels of PPARalpha and PPARgamma than normal nasal mucosa and these levels were, for PPARgamma, further reduced following steroid treatment. PPARgamma immunoreactivity was detected in the epithelium, but also found in smooth muscle of blood vessels, glandular acini and inflammatory cells. Quantitative evaluation of the epithelial immunostaining revealed no differences between nasal biopsies from patients with allergic rhinitis and healthy volunteers. In polyps, the PPARgamma immunoreactivity was lower than in nasal mucosa and further decreased after steroid treatment. The down-regulation of PPARgamma, in nasal polyposis but not in turbinates during symptomatic seasonal rhinitis, suggests that PPARgamma might be of importance in long standing inflammations.

Sex steroids, the insulin-like growth factor regulatory system, and aging: implications for the management of older postmenopausal women.

PubMed

Rosen, C J; Glowacki, J; Craig, W

1998-01-01

Aging is associated with profound changes in the growth hormone/insulin-like growth factor (IGF) regulatory system. These include reductions in growth hormone, IGF-I, IGFBP3, and IGFBP-5 and an increase in IGFBP-4. These changes, coupled with rather marked declines in sex steroid production from both the ovary and adrenals may combine to have very deleterious effects on several organ systems in the postmenopausal woman. In particular, the prevalence of two very common diseases, osteoporosis and coronary artery disease, increase dramatically after the cessation of gonadal steroid production. The complex interrelationship between the IGF regulatory system and estrogens/androgens in the postmenopausal period may provide important clues as to the pathophysiology of both these disorders. In this paper, we begin to define the role of IGF-I (and its constituent IGF binding proteins) in skeletal and vascular tissue. Recent experimental data show the effects of estrogen on circulating and tissue IGFs in older individuals. Finally, estrogen replacement therapy affects the IGF regulatory system in postmenopausal women. Although conclusions from early studies remain somewhat preliminary, it is likely that the IGF regulatory system will be a prime target for future studies into the pathogenesis of several age and sex hormone related degenerative disorders.

17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae.

PubMed

Blomquist, Charles H; Lima, P H; Tarrant, A M; Atkinson, M J; Atkinson, S

2006-04-01

Steroid metabolism studies have yielded evidence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17beta-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17Beta-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17beta-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E2) and NADP+. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E2 and NADP+. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP+/NAD+ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17beta-HSD and are consistent with the presence of more than one isoform of the enzyme.

Neuroactive Steroids: Receptor Interactions and Responses

PubMed Central

Tuem, Kald Beshir; Atey, Tesfay Mehari

2017-01-01

Neuroactive steroids (NASs) are naturally occurring steroids, which are synthesized centrally as de novo from cholesterol and are classified as pregnane, androstane, and sulfated neurosteroids (NSs). NASs modulate many processes via interacting with gamma-aminobutyric acid (GABA), N-methyl-d-aspartate, serotonin, voltage-gated calcium channels, voltage-dependent anion channels, α-adrenoreceptors, X-receptors of the liver, transient receptor potential channels, microtubule-associated protein 2, neurotrophin nerve growth factor, and σ1 receptors. Among these, NSs (especially allopregnanolone) have high potency and extensive GABA-A receptors and hence demonstrate anticonvulsant, anesthetic, central cytoprotectant, and baroreflex inhibitory effects. NSs are also involved in mood and learning via serotonin and anti-nociceptive activity via T-type voltage-gated Ca2+ channels. Moreover, they are modulators of mitochondrial function, synaptic plasticity, or regulators of apoptosis, which have a role in neuroprotective via voltage-dependent anion channels receptors. For proper functioning, NASs need to be in their normal level, whereas excess and deficiency may lead to abnormalities. When they are below the normal, NSs could have a part in development of depression, neuro-inflammation, multiple sclerosis, experimental autoimmune encephalitis, epilepsy, and schizophrenia. On the other hand, stress and attention deficit disorder could occur during excessive level. Overall, NASs are very important molecules with major neuropsychiatric activity. PMID:28894435

[Steroid hormones and pancreas: a new paradigm].

PubMed

Morales-Miranda, Angélica; Robles-Díaz, Guillermo; Díaz-Sánchez, Vicente

2007-01-01

The relation between steroid hormones and pancreatic function has been poorly discussed and not very well understood. In general, there is a lack of recognition among the scientific community about the importance of steroids in pancreatic function (current paradigm). In the present article we present basic, as well as clinic and epidemiologic data that demonstrate steroid synthesis and steroid biotransformation by pancreatic tissue, how exocrine and endocrine functions are modulated by steroids, the gender specific frequency and behavior of some tumors and the use of synthetic steroids and steroid action antagonists as therapeutic agents. With the available information it is possible to establish that: 1. Pancreatic tissue synthesize and transform steroid hormones. 2. Pancreatic tissue respond to steroid hormones and express steroid specific receptor molecules. 3. Some endocrine functions such as insulin synthesis and release are modulated by steroids. 4. Tumor growth is modulated by steroids and anti-steroid drugs. This set of data creates a new paradigm for the holistic study of pancreas and opens new research fields. The application of this new paradigm might result in an increase in the knowledge of pancreatic physiology, in the design of new and better diagnostic methods and eventually in the design of more effective medical treatments for the pancreatic cancers.

Assessing the links among environmental contaminants, endocrinology, and parasites to understand amphibian declines in montane regions of Costa Rica

PubMed Central

Ralicki, Hannah F.; Laurencio, David; Crocker-Buta, Sarah

2018-01-01

Amphibians inhabiting montane riparian zones in the Neotropics are particularly vulnerable to decline, but the reasons are poorly understood. Because environmental contaminants, endocrine disruption, and pathogens often figure prominently in amphibian declines it is imperative that we understand how these factors are potentially interrelated to affect montane populations. One possibility is that increased precipitation associated with global warming promotes the deposition of contaminants in montane regions. Increased exposure to contaminants, in turn, potentially elicits chronic elevations in circulating stress hormones that could contribute to montane population declines by compromising resistance to pathogens and/or production of sex steroids regulating reproduction. Here, we test this hypothesis by examining contaminant levels, stress and sex steroid levels, and nematode abundances in male drab treefrogs, Smilisca sordida, from lowland and montane populations in Costa Rica. We found no evidence that montane populations were more likely to possess contaminants (i.e., organochlorine, organophosphate and carbamate pesticides or benzidine and chlorophenoxy herbicides) than lowland populations. We also found no evidence of elevational differences in circulating levels of the stress hormone corticosterone, estradiol or progesterone. However, montane populations possessed lower androgen levels, hosted more nematode species, and had higher nematode abundances than lowland populations. Although these results suggested that nematodes contributed to lower androgens in montane populations, we were unable to detect a significant inverse relationship between nematode abundance and androgen level. Our results suggest that montane populations of this species are not at greater risk of exposure to contaminants or chronic stress, but implicate nematodes and compromised sex steroid levels as potential threats to montane populations. PMID:29324824

Does BPA alter steroid hormone synthesis in human granulosa cells in vitro?

PubMed

Mansur, Abdallah; Adir, Michal; Yerushalmi, Gil; Hourvitz, Ariel; Gitman, Hila; Yung, Yuval; Orvieto, Raoul; Machtinger, Ronit

2016-07-01

Does Bisphenol A (BPA) impair steroid hormone production in human luteinized granulosa cells in vitro? At supra-physiological concentrations, BPA alters progesterone and estradiol synthesis in vitro and significantly reduces the mRNA and protein expression levels of three genes encoding steroidogenesis enzymes. In IVF patients, the effects of BPA exposure on cycle outcome are controversial. Previous animal studies have shown that granulosa cell steroid hormone synthesis is compromised after BPA exposure, but their findings have been difficult to replicate in humans due, in part, to the low availability of discarded biological material. Luteinized granulosa cells obtained from 44 fertile and infertile patients undergoing in vitro fertilization were cultured for 48 h with different concentrations of BPA (0, 0.2, 0.02, 2.0, 20 µg/ml). Culture medium and total RNA extracted from the luteinized granulosa cells were examined for estradiol and progesterone levels as well as mRNA and protein expression of steroidogenesis enzymes, using enzyme immunoassays, real-time PCR and western blots. Treatment of granulosa cells with 2 or 20 µg/ml BPA for 48 h resulted in significantly lower progesterone biosynthesis (P < 0.005 and <0.001, respectively). Estradiol production was significantly altered only after incubation with 20 µg/ml of BPA (P < 0.001). These concentrations also significantly reduced the mRNA levels of 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP11A1 and CYP19A1 without affecting StAR and 17β-hydroxysteroid dehydrogenase mRNA expression. Similarly, 3β-HSD, CYP11A1 and CYP19A1 protein levels were reduced after administration of 20 µg/ml BPA. Lower BPA concentrations similar to, and up to 100 times, the concentrations measured in human follicular fluid, serum and urine did not alter steroidogenesis in primary granulosa cell cultures. This was an in vitro study investigating the effects of acute exposure (48 h) of BPA on discarded material. As such, the model may not accurately reflect the effect of BPA on the physiological events of follicular steroid hormone synthesis in vivo. Our results show that in vitro exposure to BPA at low doses does not affect granulosa cells steroidogenesis. Combined with recent in vivo studies, these data can be reassuring but further studies are needed to assess the effects of chronic exposure to BPA on ovarian steroidogenesis. This study was supported by grant number 1936/12 from the Israeli Science Foundation (ISF). The authors have no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Steroids

MedlinePlus

... Staying Safe Videos for Educators Search English Español Steroids KidsHealth / For Kids / Steroids What's in this article? ... a good idea to avoid them. What Are Steroids? "Steroids" has more than one meaning. Your body ...

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions

PubMed Central

Shu, H-J; Zeng, C-M; Wang, C; Covey, D F; Zorumski, C F; Mennerick, S

2006-01-01

Background and purpose: Neuroactive steroids are potent modulators of GABAA receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABAA receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABAA receptor, such as (3α,5α)-3-hydroxypregnan-20-one (3α5αP, allopregnanolone). Experimental approach: To assess the versatility of cyclodextrins as steroid modulators, we investigated interactions between γ-cyclodextrin and neuroactive steroids of different structural classes. Key results: Both a bioassay based on electrophysiological assessment of GABAA receptor function and optical measurements of cellular accumulation of a fluorescent steroid analogue suggest that γ-cyclodextrin sequesters steroids rather than directly influencing GABAA receptor function. Neither a 5β-reduced A/B ring fusion nor a sulphate group at carbon 3 affected the presumed inclusion complex formation between steroid and γ-cyclodextrin. Apparent dissociation constants for interactions between natural steroids and γ-cyclodexrin ranged from 10-60 μM. Although γ-cyclodextrin accommodates a range of natural and synthetic steroids, C11 substitutions reduced inclusion complex formation. Using γ-cyclodextrin to remove steroid not directly bound to GABAA receptors, we found that cellular retention of receptor-unbound steroid rate limits potentiation by 3α- hydroxysteroids but not inhibition by sulphated steroids. Conclusions and implications: We conclude that γ-cyclodextrins can be useful, albeit non-specific, tools for terminating the actions of multiple classes of naturally occurring neuroactive steroids. PMID:17160009

Treatment of Fragile X Syndrome with a Neuroactive Steroid

DTIC Science & Technology

2012-08-01

common form of inherited intellectual disability and autism . The protein (FMRP) encoded by the fragile X mental retardation gene (FMR1), is an RNA...FXS) is the most common inherited cause of mental retardation, and the most common single gene mutation associated with autism (Demark et al. 2003...profile of impairments, with the most interesting being comorbidity with autism . From the most recent studies, the prevalence of autism spectrum

Pushing quantitation limits in micro UHPLC-MS/MS analysis of steroid hormones by sample dilution using high volume injection.

PubMed

Márta, Zoltán; Bobály, Balázs; Fekete, Jenő; Magda, Balázs; Imre, Tímea; Mészáros, Katalin Viola; Szabó, Pál Tamás

2016-09-10

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LoQ). Micro UHPLC coupling with sensitive tandem mass spectrometry provides state of the art solutions for such analytical problems. Decreased column volume in micro LC limits the injectable sample volume. However, if analyte concentration is extremely low, it might be necessary to inject high sample volumes. This is particularly critical for strong sample solvents and weakly retained analytes, which are often the case when preparing biological samples (protein precipitation, sample extraction, etc.). In that case, high injection volumes may cause band broadening, peak distortion or even elution in dead volume. In this study, we evaluated possibilities of high volume injection onto microbore RP-LC columns, when sample solvent is diluted. The presented micro RP-LC-MS/MS method was optimized for the analysis of steroid hormones from human plasma after protein precipitation with organic solvents. A proper sample dilution procedure helps to increase the injection volume without compromising peak shapes. Finally, due to increased injection volume, the limit of quantitation can be decreased by a factor of 2-5, depending on the analytes and the experimental conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Dexamethasone-induced haptoglobin release by calf liver parenchymal cells.

PubMed

Higuchi, H; Katoh, N; Miyamoto, T; Uchida, E; Yuasa, A; Takahashi, K

1994-08-01

Parenchymal cells were isolated from the liver of male calves, and monolayer cultures formed were treated with glucocorticoids to examine whether haptoglobin, appearance of which is associated with hepatic lipidosis (fatty liver) in cattle, is induced by steroid hormones. Without addition of dexamethasone, only trace amounts of haptoglobin were detected in culture medium. With addition of dexamethasone (10(-12) to 10(-4) M), considerable amounts of haptoglobin were released into the medium. Maximal release was observed at concentrations of 10(-8) to 10(-6) M dexamethasone. Haptoglobin release was similarly induced by cortisol, although the effect was less potent than that of dexamethasone. Actinomycin D (a known protein synthesis inhibitor) dose-dependently reduced amounts of haptoglobin released in response to 10(-8) M dexamethasone. Dexamethasone also induced annexin I, which is known to be synthesized in response to glucocorticoids. Dexamethasone treatment resulted in reduced protein kinase C activity in the cell cytosol, which has been shown to be an early event in dexamethasone-treated cells. Other than glucocorticoids, estradiol induced haptoglobin release, whereas progesterone was less effective. The association of haptoglobin with hepatic lipidosis can be reasonably explained by the fact that haptoglobin production by the liver is induced by glucocorticoids and estradiol, and these steroid hormones are triggers for development of hepatic lipidosis in cattle.

Development of Laboratory Investigations in Disorders of Sex Development.

PubMed

Audí, Laura; Camats, Núria; Fernández-Cancio, Mónica; Granada, María L

2018-01-01

Scientific knowledge to understand the biological basis of sex development was prompted by the observation of variants different from the 2 most frequent body types, and this became one of the fields first studied by modern pediatric endocrinology. The clinical observation was supported by professionals working in different areas of laboratory sciences which led to the description of adrenal and gonadal steroidogenesis, the enzymes involved, and the different deficiencies. Steroid hormone measurements evolved from colorimetry to radioimmunoassay (RIA) and automated immunoassays, although gas and liquid chromatography coupled to mass spectrometry are now the gold standard techniques for steroid measurements. Peptide hormones and growth factors were purified, and their measurement evolved from RIA to automated immunoassays. Hormone action mechanisms were described, and their specific receptors were characterized and assayed in experimental materials and in patient tissues and cell cultures. The discovery of the genetic basis for variant sex developments began with the description of the sex chromosomes. Molecular technology allowed cloning of genes coding for the different proteins involved in sex determination and development. Experimental animal models aided in verifying the roles of proteins and also suggested new genes to be investigated. New candidate genes continue to be described based on experimental models and on next-generation sequencing of patient DNAs. © 2017 S. Karger AG, Basel.

Secretion of angiogenic proteins by human multipotent mesenchymal stromal cells and their clinical potential in the treatment of avascular osteonecrosis.

PubMed

Müller, I; Vaegler, M; Holzwarth, C; Tzaribatchev, N; Pfister, S M; Schütt, B; Reize, P; Greil, J; Handgretinger, R; Rudert, M

2008-11-01

Osteonecrosis is a frequent complication after treatment for childhood leukemia and other steroid-based therapies. The success rate of core decompression surgery is limited. Therefore, we evaluated relevant biological characteristics of human multipotent mesenchymal stromal cells (MSCs) in vitro. MSCs cultured under low-oxygen tensions showed decreased proliferation and differentiation into bone. However, these MSCs secreted significant amounts of vascular endothelial-derived factor in the presence of interferon-gamma. These in vitro results with potential effects on neovascularization and bone regeneration as well as findings in animal models prompted us to treat five patients with steroid-induced osteonecrosis of the femur by core decompression surgery and instillation of expanded autologous MSCs. Within 3 weeks of culture, sufficient numbers of MSCs were generated using animal protein-free culture conditions. No chromosomal aberrations were detected by matrix-based comparative genomic hybridization. Application of MSCs during core decompression was feasible and safe. Median follow-up is 16 months and the patients in this pilot study reported clinical improvement. Formation of mineralized bone in the osteonecrotic cavity was proven by computed tomography. Taken together, MSCs display biological properties that may add to the efficiency of surgical treatment in osteonecrosis and should be evaluated in larger patient cohorts.

Immune-mediated steroid-responsive epileptic spasms and epileptic encephalopathy associated with VGKC-complex antibodies.

PubMed

Suleiman, Jehan; Brenner, Tanja; Gill, Deepak; Troedson, Christopher; Sinclair, Adriane J; Brilot, Fabienne; Vincent, Angela; Lang, Bethan; Dale, Russell C

2011-11-01

Autoantibodies that bind to voltage-gated potassium-channel complex proteins (VGKC-complex antibodies) occur frequently in adults with limbic encephalitis presenting with cognitive impairment and seizures. Recently, VGKC-complex antibodies have been described in a few children with limbic encephalitis, and children with unexplained encephalitis presenting with status epilepticus. We report a case of infantile-onset epileptic spasms and developmental delay compatible with epileptic encephalopathy. Our patient was a female infant, aged 4 months at presentation. She had evidence of immune activation in the central nervous system with elevated cerebrospinal fluid neopterin and mirrored oligoclonal bands, which prompted testing for autoantibodies. VGKC-complex antibodies were elevated (201 pmol/L, normal<100), but extended antibody testing, including leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein 2 (CASPR2), was negative. The patient showed a partial response to steroid treatment, which was started late in the disease course. On review at 13 months of age, her development was consistent with an age of 5 to 6 months. These results suggest that VGKC-complex antibodies might represent a marker of immune therapy responsiveness in a subgroup of patients with infantile epileptic encephalopathy. © The Authors. Developmental Medicine & Child Neurology © 2011 Mac Keith Press.

Endocrine-immune interactions in human endometrium.

PubMed

Kayisli, U A; Guzeloglu-Kayisli, O; Arici, A

2004-12-01

The immune system is a complex entity designed to eliminate foreign intruding antigens and is influenced by and, in turn, influences the function of the reproductive system. Despite the widespread associations between immunology and reproductive medicine, the study of system interactions remains in its infancy. Many diverse facts are accumulating, and pieces of the puzzle are becoming available to provide a clearer picture. In this review article, we focus on the interactions between endocrine and immune systems in the human endometrium. Understanding the molecular pathways in endocrine-immune interactions in the human endometrium is crucial to understand events such as menstrual bleeding, tissue repair and regeneration, inflammation, angiogenesis, blastocyst implantation, and progression of pregnancy. These events require a balanced regulation of endometrial differentiation, proliferation, cell survival, leukocyte recruitment, apoptosis, and angiogenesis by sex steroids. In this review, we first outline the role of survival factors such as phosphoinositol 3-kinase/protein kinase B, PTEN, NFkappaB, and apoptotic molecules (Fas-FasL, Bcl-2). We then discuss their regulation by estrogen and progesterone in the endometrium. We present evidence for direct and/or indirect roles of steroid hormones on the expression of chemotactic cytokines (interleukin-8 and monocyte chemotactic protein-1) and on the survival versus apoptosis of resident endometrial cells (stromal, epithelial, and endothelial cells) and nonresident cells (leukocytes).

Yeast metabolic engineering--targeting sterol metabolism and terpenoid formation.

PubMed

Wriessnegger, Tamara; Pichler, Harald

2013-07-01

Terpenoids comprise various structures conferring versatile functions to eukaryotes, for example in the form of prenyl-anchors they attach proteins to membranes. The physiology of eukaryotic membranes is fine-tuned by another terpenoid class, namely sterols. Evidence is accumulating that numerous membrane proteins require specific sterol structural features for function. Moreover, sterols are intermediates in the synthesis of steroids serving as hormones in higher eukaryotes. Like steroids many compounds of the terpenoid family do not contribute to membrane architecture, but serve as signalling, protective or attractant/repellent molecules. Particularly plants have developed a plenitude of terpenoid biosynthetic routes branching off early in the sterol biosynthesis pathway and, thereby, forming one of the largest groups of naturally occurring organic compounds. Many of these aromatic and volatile molecules are interesting for industrial application ranging from foods to pharmaceuticals. Combining the fortunate situation that sterol biosynthesis is highly conserved in eukaryotes with the amenability of yeasts to genetic and metabolic engineering, basically all naturally occurring terpenoids might be produced involving yeasts. Such engineered yeasts are useful for the study of biological functions and molecular interactions of terpenoids as well as for the large-scale production of high-value compounds, which are unavailable in sufficient amounts from natural sources due to their low abundance. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hormonal regulation of growth and life span of bullfrog tadpole tail epidermal cells cultured in vitro.

PubMed

Nishikawa, A; Yoshizato, K

1986-02-01

Epidermal cells were dissociated from tails of the bullfrog tadpole, Rana catesbeiana, and cultured to investigate their response to steroid and thyroid hormones. Charcoal-treated serum (CTS) was used in the growth medium when cells were to be grown in the absence of steroid and thyroid hormones. The cells could be maintained for 2 weeks with a small increase in cell number in medium that contained CTS (CTS medium). Addition of cortisol to CTS medium increased both cellular attachment to the culture dishes and the proliferation of the attached cells with an optimum concentration of 5 X 10(-7) M. The cells remained viable and attached for at least a week. Cortisol stimulated the rate of protein synthesis 1.8-fold but did not alter the rate of DNA synthesis. The cells did not proliferate in the medium containing triiodothyronine (T3) and detached themselves from the dish within 5 days, which occurred in a dose-dependent manner with a maximum effect at 10(-8) M. It drastically decreased the rate of DNA synthesis but did not influence the rate of protein synthesis. These responses of cells to cortisol and T3 may reflect growth and death of tail epidermal cells in vivo at metamorphosis.

Selective androgen receptor modulators as function promoting therapies.

PubMed

Bhasin, Shalender; Jasuja, Ravi

2009-05-01

The past decade has witnessed an unprecedented discovery effort to develop selective androgen receptor modulators (SARMs) that improve physical function and bone health without adversely affecting the prostate and cardiovascular outcomes. This review describes the historical evolution, the rationale for SARM development, and the mechanisms of testosterone action and SARM selectivity. Although steroidal SARMs have been around since the 1940s, a number of nonsteroidal SARMs that do not serve as substrates for CYP19 aromatase or 5alpha-reductase, act as full agonists in muscle and bone and as partial agonists in prostate are in development. The differing interactions of steroidal and nonsteroidal compounds with androgen receptor (AR) contribute to their unique pharmacologic actions. Ligand binding induces specific conformational changes in the ligand-binding domain, which could modulate surface topology and protein-protein interactions between AR and coregulators, resulting in tissue-specific gene regulation. Preclinical studies have demonstrated the ability of SARMs to increase muscle and bone mass in preclinical rodent models with varying degree of prostate sparing. Phase I trials of SARMs in humans have reported modest increments in fat-free mass. SARMs hold promise as a new class of function promoting anabolic therapies for a number of clinical indications, including functional limitations associated with aging and chronic disease, frailty, cancer cachexia, and osteoporosis.

Selective Androgen Receptor Modulators (SARMs) as Function Promoting Therapies

PubMed Central

Bhasin, Shalender; Jasuja, Ravi

2010-01-01

Purpose of review The last decade has witnessed unprecedented discovery effort to develop selective androgen receptor modulators (SARMs) that improve physical function and bone health without adversely affecting the prostate and cardiovascular outcomes. This review describes the historical evolution, the rationale for SARM development, and the mechanisms of testosterone action and SARM selectivity. Recent Findings While steroidal SARMs have been around since the 1940s, a number of nonsteroidal SARMs that do not serve as substrates for CYP19 aromatase or 5α-reductase, act as full agonists in muscle and bone and as partial agonists in prostate are in development. The differing interactions of steroidal and nonsteroidal compounds with AR contribute to their unique pharmacologic actions. Ligand binding induces specific conformational changes in the ligand binding domain, which could modulate surface topology and protein-protein interactions between AR and coregulators, resulting in tissue-specific gene regulation. Preclinical studies have demonstrated the ability of SARMs to increase muscle and bone mass in preclinical rodent models with varying degree of prostate sparing. Phase I trials of SARMs in humans have reported modest increments in fat-free mass. Summary SARMs hold promise as a new class of function promoting anabolic therapies for a number of clinical indications, including functional limitations associated with aging and chronic disease, frailty, cancer cachexia, and osteoporosis. PMID:19357508

Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

PubMed Central

Yao, Hongjie; Brick, Kevin; Evrard, Yvonne; Xiao, Tiaojiang; Camerini-Otero, R. Daniel; Felsenfeld, Gary

2010-01-01

CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function. PMID:20966046

Possible role of the aromatase-independent steroid metabolism pathways in hormone responsive primary breast cancers.

PubMed

Hanamura, Toru; Niwa, Toshifumi; Gohno, Tatsuyuki; Kurosumi, Masafumi; Takei, Hiroyuki; Yamaguchi, Yuri; Ito, Ken-ichi; Hayashi, Shin-ichi

2014-01-01

Aromatase inhibitors (AIs) exert antiproliferative effects by reducing local estrogen production from androgens in postmenopausal women with hormone-responsive breast cancer. Previous reports have shown that androgen metabolites generated by the aromatase-independent enzymes, 5α-androstane-3β, 17β-diol (3β-diol), androst-5-ene-3β, and 17β-diol (A-diol), also activate estrogen receptor (ER) α. Estradiol (E2) can also reportedly be generated from estrone sulfate (E1S) pooled in the plasma. Estrogenic steroid-producing aromatase-independent pathways have thus been proposed as a mechanism of AI resistance. However, it is unclear whether these pathways are functional in clinical breast cancer. To investigate this issue, we assessed the transcriptional activities of ER in 45 ER-positive human breast cancers using the adenovirus estrogen-response element-green fluorescent protein assay and mRNA expression levels of the ER target gene, progesterone receptor, as indicators of ex vivo and in vivo ER activity, respectively. We also determined mRNA expression levels of 5α-reductase type 1 (SRD5A1) and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD type 1; HSD3B1), which produce 3β-diol from androgens, and of steroid sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD type 1; HSD17B1), which produce E2 or A-diol from E1S or dehydroepiandrosterone sulfate. SRD5A1 and HSD3B1 expression levels were positively correlated with ex vivo and in vivo ER activities. STS and HSD17B1 expression levels were positively correlated with in vivo ER activity alone. Elevated expression levels of these steroid-metabolizing enzymes in association with high in vivo ER activity were particularly notable in postmenopausal patients. Analysis of the expression levels of steroid-metabolizing enzymes revealed positive correlations between SRD5A1 and HSD3B1, and STS and HSD17B1. These findings suggest that the SRD5A1-HSD3B1 as well as the STS-HSD17B pathways, could contributes to ER activation, especially postmenopause. These pathways might function as an alternative estrogenic steroid-producing, aromatase-independent pathways.

Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Jinmei; Wang Xuefeng; Xi Zhiqin

2006-10-06

Purpose: TRAP220 (thyroid hormone receptor-associated protein) functions as a coactivator for nuclear receptors and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. Thus, TRAP220 enhances the function of thyroid/steroid hormone receptors such as thyroid hormone and oestrogen receptors. This study investigated the expression of TRAP220 mRNA and protein level in epileptic brains comparing with human control. Methods: We examined the expression of TRAP220 mRNA and protein levels in temporal lobes from patients with chronic pharmacoresistant epilepsy who have undergone surgery. Results: Expression of TRAP220 mRNA and protein was shown to be decreased significantly in themore » temporal cortex of the patients with epilepsy. Conclusions: Our work showed that a decrease in TRAP220 mRNA and protein levels may be involved in the pathophysiology of epilepsy and may be associated with impairment of the brain caused by frequent seizures.« less

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells

PubMed Central

Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase. PMID:27428326

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

PubMed

Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase.

Deletion of flbA results in increased secretome complexity and reduced secretion heterogeneity in colonies of Aspergillus niger.

PubMed

Krijgsheld, Pauline; Nitsche, Benjamin M; Post, Harm; Levin, Ana M; Müller, Wally H; Heck, Albert J R; Ram, Arthur F J; Altelaar, A F Maarten; Wösten, Han A B

2013-04-05

Aspergillus niger is a cell factory for the production of enzymes. This fungus secretes proteins in the central part and at the periphery of the colony. The sporulating zone of the colony overlapped with the nonsecreting subperipheral zone, indicating that sporulation inhibits protein secretion. Indeed, strain ΔflbA that is affected early in the sporulation program secreted proteins throughout the colony. In contrast, the ΔbrlA strain that initiates but not completes sporulation did not show altered spatial secretion. The secretome of 5 concentric zones of xylose-grown ΔflbA colonies was assessed by quantitative proteomics. In total 138 proteins with a signal sequence for secretion were identified in the medium of ΔflbA colonies. Of these, 18 proteins had never been reported to be part of the secretome of A. niger, while 101 proteins had previously not been identified in the culture medium of xylose-grown wild type colonies. Taken together, inactivation of flbA results in spatial changes in secretion and in a more complex secretome. The latter may be explained by the fact that strain ΔflbA has a thinner cell wall compared to the wild type, enabling efficient release of proteins. These results are of interest to improve A. niger as a cell factory.

Active zone density is conserved during synaptic growth but impaired in aged mice

PubMed Central

Chen, Jie; Mizushige, Takafumi; Nishimune, Hiroshi

2013-01-01

Presynaptic active zones are essential structures for synaptic vesicle release, but the developmental regulation of their number and maintenance during aging at mammalian neuromuscular junctions (NMJs) remains unknown. Here, we analyzed the distribution of active zones in developing, mature, and aged mouse NMJs by immunohistochemical detection of the active zone-specific protein Bassoon. Bassoon is a cytosolic scaffolding protein essential for the active zone assembly in ribbon synapses and some brain synapses. Bassoon staining showed a punctate pattern in nerve terminals and axons at the nascent NMJ on embryonic days 16.5–18.5. Three-dimensional reconstruction of NMJs revealed that the majority of Bassoon puncta within an NMJ were attached to the presynaptic membrane from postnatal day 0 to adulthood, and colocalized with another active zone protein Piccolo. During postnatal development, the number of Bassoon puncta increased as the size of the synapses increased. Importantly, the density of Bassoon puncta remained relatively constant from postnatal day 0 to 54 at 2.3 puncta/μm2, while the synapse size increased 3.3-fold. However, Bassoon puncta density and signal intensity were significantly attenuated at the NMJs of 27-month-old aged mice. These results suggest that synapses maintain the density of synaptic vesicle release sites while the synapse size changes, but this density becomes impaired during aging. PMID:21935939

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

PubMed Central

Barski, Oleg A.; Tipparaju, Srinivas M.; Bhatnagar, Aruni

2008-01-01

The Aldo-Keto Reductase (AKR) superfamily comprises of several enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism and detoxification. Substrates of the family include glucose, steroids, glycosylation end products, lipid peroxidation products, and environmental pollutants. These proteins adopt a (β/α)8 barrel structural motif interrupted by a number of extraneous loops and helixes that vary between proteins and bring structural identity to individual families. The human AKR family differs from the rodent families. Due to their broad substrate specificity, AKRs play an important role in the Phase II detoxification of a large number of pharmaceuticals, drugs, and xenobiotics. PMID:18949601

Optimised deconjugation of androgenic steroid conjugates in bovine urine.

PubMed

Pedersen, Mikael; Frandsen, Henrik L; Andersen, Jens H

2017-04-01

After administration of steroids to animals the steroids are partially metabolised in the liver and kidney to phase 2 metabolites, i.e., glucuronic acid or sulphate conjugates. During analysis these conjugated metabolites are normally deconjugated enzymatically with aryl sulphatase and glucuronidase resulting in free steroids in the extract. It is well known that some sulphates are not deconjugated using aryl sulphatase; instead, for example, solvolysis can be used for deconjugation of these aliphatic sulphates. The effectiveness of solvolysis on androgenic steroid sulphates was tested with selected aliphatic steroid sulphates (boldenone sulphate, nortestosteron sulphate and testosterone sulphate), and the method was validated for analysis of androgenic steroids in bovine urine using free steroids, steroid sulphates and steroid glucuronides as standards. Glucuronidase and sulphuric acid in ethyl acetate were used for deconjugation and the extract was purified by solid-phase extraction. The final extract was evaporated to dryness, re-dissolved and analysed by LC-MS/MS.

Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

PubMed

Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

2015-06-15

High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Progesterone protects normative anxiety-like responding among ovariectomized female mice that conditionally express the HIV-1 regulatory protein, Tat, in the CNS.

PubMed

Paris, Jason J; Fenwick, Jason; McLaughlin, Jay P

2014-05-01

Increased anxiety is co-morbid with human immunodeficiency virus (HIV) infection. Actions of the neurotoxic HIV-1 regulatory protein, Tat, may contribute to affective dysfunction. We hypothesized that Tat expression would increase anxiety-like behavior of female GT-tg bigenic mice that express HIV-1 Tat protein in the brain in a doxycycline-dependent manner. Furthermore, given reports that HIV-induced anxiety may occur at lower rates among women, and that the neurotoxic effects of Tat are ameliorated by sex steroids in vitro, we hypothesized that 17β-estradiol and/or progesterone would ameliorate Tat-induced anxiety-like effects. Among naturally-cycling proestrous and diestrous mice, Tat-induction via 7days of doxycycline treatment significantly increased anxiety-like responding in an open field, elevated plus maze and a marble-burying task, compared to treatment with saline. Proestrous mice demonstrated less anxiety-like behavior than diestrous mice in the open field and elevated plus maze, but these effects did not significantly interact with Tat-induction. Among ovariectomized mice, doxycycline-induced Tat protein significantly increased anxiety-like behavior in an elevated plus maze and a marble burying task compared to saline-treated mice, but not an open field (where anxiety-like responding was already maximal). Co-administration of progesterone (4mg/kg), but not 17β-estradiol (0.09mg/kg), with doxycycline significantly ameliorated anxiety-like responding in the elevated plus maze and marble burying tasks. When administered together, 17β-estradiol partially antagonized the protective effects of progesterone on Tat-induced anxiety-like behavior. These findings support evidence of steroid-protection over HIV-1 proteins, and extend them by demonstrating the protective capacity of progesterone on Tat-induced anxiety-like behavior of ovariectomized female mice. Copyright © 2014 Elsevier Inc. All rights reserved.

Progesterone treatment modulates mRNA OF neurosteroidogenic enzymes in a murine model of multiple sclerosis.

PubMed

Garay, Laura; Gonzalez Giqueaux, Paula; Guennoun, Rachida; Schumacher, Michael; Gonzalez Deniselle, Maria Claudia; De Nicola, Alejandro F

2017-01-01

Previous studies of experimental autoimmune encephalomyelitis (EAE) have shown that progesterone decreases inflammatory cell infiltration and proinflammatory factors, increases myelination and attenuates clinical grade of EAE mice. To elucidate potential mediators of these effects, we analyzed the mRNA expression of neurosteroidogenic enzymes in the spinal cord, in view of the protective role of steroids in EAE. We also analyzed mitochondrial morphology and dynamics (fusion and fission proteins), considering the role of mitochondria in neurosteroidogenesis. EAE was induced in C57Bl6 mice using MOG 40-54 and killed on day 16 after induction. Using qPCR, we found in steroid-untreated EAE mice decreased mRNAs for the steroidogenic acute regulatory protein (Star), voltage-dependent anion channel (VDAC), P450scc (cholesterol side-chain cleavage), 5α-reductase, 3α-hydroxysteroid dehydrogenase (3α-HSD) and aromatase, whereas levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) showed a large intra-group variance. We also found increased mRNA expression of 18Kd translocator protein (TSPO), which likely resulted from the reactive microgliosis in this model. EAE mice also showed pathological mitochondrial morphology and reduced expression of fission and fusion protein mRNAs. Most importantly, pretreatment with progesterone a week before EAE induction increased Star,VDAC, P450scc, 5α-reductase type I, 3α-HSD and aromatase mRNAs and did not modify 3β-HSD. TSPO mRNA was decreased, consequent with the inhibition of microgliosis. Mitochondrial morphology was improved and fission/fusion protein mRNAs were enhanced by progesterone treatment. Furthermore, progesterone protective effects on mitochondrial and endoplasmic reticulum may allow the recovery of neurosteroidogenesis. In this way, endogenously synthesized neurosteroids may reinforce the beneficial effects of exogenous progesterone previously shown in MS mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Glucocorticoids enhance activation of the human type II 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase gene.

PubMed

Feltus, F Alex; Cote, Stephanie; Simard, Jacques; Gingras, Sebastien; Kovacs, William J; Nicholson, Wendell E; Clark, Barbara J; Melner, Michael H

2002-09-01

Glucocorticoids indirectly alter adrenocortical steroid output through the inhibition of ACTH secretion by the anterior pituitary. However, previous studies suggest that glucocorticoids can directly affect adrenocortical steroid production. Therefore, we have investigated the ability of glucocorticoids to affect transcription of adrenocortical steroid biosynthetic enzymes. One potential target of glucocorticoid action in the adrenal is an enzyme critical for adrenocortical steroid production: 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Treatment of the adrenocortical cell line (H295R) with the glucocorticoid agonist dexamethasone (DEX) increased cortisol production and 3beta-HSD mRNA levels alone or in conjunction with phorbol ester. This increase in 3beta-HSD mRNA was paralleled by increases in Steroidogenic Acute Regulatory Protein (StAR) mRNA levels. The human type II 3beta-HSD promoter lacks a consensus palindromic glucocorticoid response element (GRE) but does contain a Stat5 response element (Stat5RE) suggesting that glucocorticoids could affect type II 3beta-HSD transcription via interaction with Stat5. Transfection experiments show enhancement of human type II 3beta-HSD promoter activity by coexpression of the glucocorticoid receptor (GR) and Stat5A and treatment with 100nM dexamethasone. Furthermore, removal of the Stat5RE either by truncation of the 5' flanking sequence in the promoter or introduction of point mutations to the Stat5RE abolished the ability of DEX to enhance 3beta-HSD promoter activity. These studies demonstrate the ability of glucocorticoids to directly enhance the expression of an adrenal steroidogenic enzyme gene albeit independent of a consensus palindromic glucocorticoid response element.

Growth Pattern in Paediatric Crohn Disease Is Related to Inflammatory Status.

PubMed

Ley, Delphine; Duhamel, Alain; Behal, Hélène; Vasseur, Francis; Sarter, Hélène; Michaud, Laurent; Gower-Rousseau, Corinne; Turck, Dominique

2016-12-01

The respective role of disease activity and steroid therapy in growth impairment in paediatric-onset Crohn disease (CD) is still debated. Our aim was to investigate whether the growth pattern of children with CD was correlated with the inflammatory status during the disease course, regardless the cumulative duration of steroid therapy. One hundred and seven patients with a diagnosis of CD <17 years, followed during ≥2 years and for whom ≥2 height measures were available during follow-up, were identified between 1998 and 2010. Height, C-reactive protein (CRP), orosomucoid, and steroid therapy duration were collected at each visit. The relationship between the evolution of growth velocity and inflammatory status during follow-up was investigated using a linear mixed model with random coefficients. Median age at diagnosis was 11.7 years (Q1-Q3: 9.8-13.5). Mean height for age (H/A) z score was 0.14 ± 1.29 at diagnosis and 0.05 ± 1.23 among the 75 patients who had reached their final height at maximal follow-up (median: 4.9 years; Q1-Q3: 3.8-6.4). Growth failure (H/A z score <-2) was present in 7 (8%) patients at diagnosis and 5 (5%) at maximal follow-up. Growth velocity was negatively correlated with the evolution of CRP (P < 0.0001) and orosomucoid (P < 0.0001) during follow-up. After adjustment for the cumulative duration of steroid therapy, these 2 correlations remained significant (CRP: P = 0.0008; orosomucoid: P < 0.0001). Children with CD with uncontrolled inflammatory status have a lower growth velocity. The inflammatory status should be kept as close to normal as possible in paediatric-onset patients with CD to optimize their growth pattern.

Spin-labeled derivatives of cardiotonic steroids as tools for characterization of the extracellular entrance to the binding site on Na+ ,K+ -ATPase.

PubMed

Guo, Jin-Hua; Jiang, Ren-Wang; Andersen, Jacob Lauwring; Esmann, Mikael; Fedosova, Natalya U

2018-04-24

The information obtained from crystallized complexes of the Na + ,K + -ATPase with cardiotonic steroids (CTS) is not sufficient to explain differences in the inhibitory properties of CTS such as stereoselectivity of CTS binding or effect of glycosylation on the preference to enzyme isoforms. The uncertainty is related to the spatial organization of the hydrophilic cavity at the entrance of the CTS-binding site. Therefore, there is a need to supplement the crystallographic description with data obtained in aqueous solution, where molecules have significant degree of flexibility. This work addresses the applicability of the electron paramagnetic resonance (EPR) method for the purpose. We have designed and synthesized spin-labeled compounds based on the cinobufagin steroid core. The length of the spacer arms between the steroid core and the nitroxide group determines the position of the reporting group (N-O) confined to the binding site. High affinity to Na + ,K + -ATPase is inferred from their ability to inhibit enzymatic activity. The differences between the EPR spectra in the absence and presence of high ouabain concentrations identify the signature peaks originating from the fraction of the spin labels bound within the ouabain site. The degree of perturbations of the EPR spectra depends on the length of the spacer arm. Docking of the compounds into the CTS site suggests which elements of the protein structure might be responsible for interference with the spin label (e.g., steric clashes or immobilization). Thus, the method is suitable for gathering information on the cavity leading to the CTS-binding site in Na + ,K + -ATPase in all conformations with high affinity to CTS. © 2018 Federation of European Biochemical Societies.

Immunoglobulin M nephropathy nephropathy in adults and adolescents in India: a single-center study of natural history.

PubMed

Singhai, A M; Vanikar, A V; Goplani, K R; Kanodia, K V; Patel, R D; Suthar, K S; Patel, H V; Gumber, M R; Shah, P R; Trivedi, H L

2011-01-01

Immunoglobulin M nephropathy (IgMN) is an idiopathic glomerulonephritis (GN) usually presenting clinically as steroid resistant/dependent nephrotic syndrome (NS) with pathology of mesangial proliferative GN or focal and segmental glomerulosclerosis with diffuse predominant mesangial IgM deposits. Not much information is available about its natural history. This is the first Indian study to our knowledge on IgMN in adults and adolescents. We evaluated renal biopsies performed at our center between January,'04 to September,'09. Biopsies of all adolescents and adults were evaluated for IgMN and we studied their age, gender distribution, blood pressure (BP), disease duration, steroid/immunosuppressive management and serial serum creatinine (SCr), urinary proteins, and BP values. Patients with other systemic diseases/infections and children were excluded. IgMN constituted 4.3% of 2702 adult renal biopsies. No significant gender predilection was noted. Males presented at average age of 23.1 years, females at 30 years. Steroid-dependent NS was the commonest presentation noted in 75% followed by steroid-resistant NS. Hypertension was noted in 10% patients. Mesangial proliferative GN (MePGN) was commonest histopathological finding noted in 74.4%, followed by focal segmental glomerulosclerosis (FSGS) in 16.2%, and minimal change disease (MCD) in 9.4% biopsies. Sole IgM deposits were noted in 88.5%. All MCD, 35.6% MePGN reached remission, FSGS progressed to renal failure by 1 year. Hypertension, proteinuria, interstitial fibrosis, and FSGS were bad prognosticators. This is the first Indian study of IgMN in adults and adolescents carried out over a period of 5.8 years, which has shown that hypertension, proteinuria, and interstitial fibrosis at presentation have bad prognosis.

Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basavarajappa, Mallikarjuna S., E-mail: mbasava2@illinois.edu; Craig, Zelieann R., E-mail: zelieann@illinois.edu; Hernandez-Ochoa, Isabel, E-mail: mihernandez@cinvestav.mx

2011-06-15

The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E{sub 2}) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition,more » sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E{sub 2} metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E{sub 2}, testosterone, androstenedione, and progesterone (P{sub 4}) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17{beta}-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17{alpha}-hydroxylase/17,20-lyase (Cyp17a1), 3{beta} hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels. - Highlights: > MXC inhibits steroidogenesis > MXC inhibits steroidogenic enzymes > MXC induces metabolic enzymes« less

The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase.

PubMed

Devendran, Saravanan; Mythen, Sean M; Ridlon, Jason M

2018-06-01

Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydro-xyandrostenedione. A cortisol-inducible operon ( desABCD ) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were K m of 4.96 ± 0.57 µM and k cat of 0.87 ± 0.076 min -1 Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids. Copyright © 2018 Devendran et al.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

PubMed

Schindelman, G; Morikami, A; Jung, J; Baskin, T I; Carpita, N C; Derbyshire, P; McCann, M C; Benfey, P N

2001-05-01

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis

PubMed Central

Schindelman, Gary; Morikami, Atsushi; Jung, Jee; Baskin, Tobias I.; Carpita, Nicholas C.; Derbyshire, Paul; McCann, Maureen C.; Benfey, Philip N.

2001-01-01

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion. PMID:11331607

Visualization of Synchronous or Asynchronous Release of Single Synaptic Vesicle in Active-Zone-Like Membrane Formed on Neuroligin-Coated Glass Surface.

PubMed

Funahashi, Junichiro; Tanaka, Hiromitsu; Hirano, Tomoo

2018-01-01

Fast repetitive synaptic transmission depends on efficient exocytosis and retrieval of synaptic vesicles around a presynaptic active zone. However, the functional organization of an active zone and regulatory mechanisms of exocytosis, endocytosis and reconstruction of release-competent synaptic vesicles have not been fully elucidated. By developing a novel visualization method, we attempted to identify the location of exocytosis of a single synaptic vesicle within an active zone and examined movement of synaptic vesicle protein synaptophysin (Syp) after exocytosis. Using cultured hippocampal neurons, we induced formation of active-zone-like membranes (AZLMs) directly adjacent and parallel to a glass surface coated with neuroligin, and imaged Syp fused to super-ecliptic pHluorin (Syp-SEP) after its translocation to the plasma membrane from a synaptic vesicle using total internal reflection fluorescence microscopy (TIRFM). An AZLM showed characteristic molecular and functional properties of a presynaptic active zone. It contained active zone proteins, cytomatrix at the active zone-associated structural protein (CAST), Bassoon, Piccolo, Munc13 and RIM, and showed an increase in intracellular Ca 2+ concentration upon electrical stimulation. In addition, single-pulse stimulation sometimes induced a transient increase of Syp-SEP signal followed by lateral spread in an AZLM, which was considered to reflect an exocytosis event of a single synaptic vesicle. The diffusion coefficient of Syp-SEP on the presynaptic plasma membrane after the membrane fusion was estimated to be 0.17-0.19 μm 2 /s, suggesting that Syp-SEP diffused without significant obstruction. Synchronous exocytosis just after the electrical stimulation tended to occur at multiple restricted sites within an AZLM, whereas locations of asynchronous release occurring later after the stimulation tended to be more scattered.

Marine steroids as potential anticancer drug candidates: In silico investigation in search of inhibitors of Bcl-2 and CDK-4/Cyclin D1.

PubMed

Saikia, Surovi; Kolita, Bhaskor; Dutta, Partha P; Dutta, Deep J; Neipihoi; Nath, Shyamalendu; Bordoloi, Manobjyoti; Quan, Pham Minh; Thuy, Tran Thu; Phuong, Doan Lan; Long, Pham Quoc

2015-10-01

Star fishes (Asteroidea) are rich in polar steroids with diverse structural characteristics. The structural modifications of star fish steroids occur at 3β, 4β, 5α, 6α (or β), 7α (or β), 8, 15α (or β) and 16β positions of the steroidal nucleus and in the side chain. Widely found polar steroids in starfishes include polyhydroxysteroids, steroidal sulfates, glycosides, steroid oligoglycosides etc. Bioactivity of these steroids is less studied; only a few reports like antibacterial, cytotoxic activity etc. are available. In continuation of our search for bioactive molecules from natural sources, we undertook in silico screening of steroids from star fishes against Bcl-2 and CDK-4/Cyclin D1 - two important targets of progression and proliferation of cancer cells. We have screened 182 natural steroids from star fishes occurring in different parts of the world and their 282 soft-derivatives by in silico methods. Their physico-chemical properties, drug-likeliness, binding potential with the selected targets, ADMET (absorption, distribution, metabolism, toxicity) were predicted. Further, the results were compared with those of existing steroidal and non steroidal drugs and inhibitors of Bcl-2 and CDK-4/Cyclin D1. The results are promising and unveil that some of these steroids can be potent leads for cancer treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

Long-interval Cytapheresis as a Novel Therapeutic Strategy Leading to Dosage Reduction and Discontinuation of Steroids in Steroid-dependent Ulcerative Colitis

PubMed Central

Iizuka, Masahiro; Etou, Takeshi; Kumagai, Makoto; Matsuoka, Atsushi; Numata, Yuka; Sagara, Shiho

2017-01-01

Objective This study was performed to confirm the efficacy of long-interval cytapheresis on steroid-dependent ulcerative colitis (UC). Methods To discontinue steroids in patients with steroid-dependent UC, we previously designed a novel regimen of cytapheresis (CAP), which we termed “long-interval cytapheresis (LI-CAP)”, in which CAP was performed as one session every two or three weeks and continued during the whole period of tapering steroid dosage. In this study, we performed LI-CAP therapy 20 times (11 male and 9 female; mean age 41.8 years) between April 2010 and April 2015 for 14 patients with steroid-dependent UC. We evaluated the effectiveness of LI-CAP by examining the improvement in Lichtiger's clinical activity index (CAI), the rate of clinical remission, and the rate of steroid discontinuation. We further examined the rate of sustained steroid-free clinical remission at 6 and 12 months after LI-CAP in patients who successfully discontinued steroid-use after LI-CAP. The primary endpoint was the rate of discontinuation of steroids after LI-CAP. Results The mean CAI score before LI-CAP (7.550) significantly decreased to 1.65 after LI-CAP (p<0.0001). The rate of clinical remission after LI-CAP was 80%. The rate of steroid discontinuation after LI-CAP was 60.0%. The mean dose of daily prednisolone was significantly decreased after LI-CAP (2.30 mg) compared with that before therapy (17.30 mg) (p=0.0003). The rate of sustained steroid-free clinical remission after LI-CAP was 66.7% at 6 months and 66.7% at 12 months. Conclusion We confirmed that LI-CAP has therapeutic effects on reducing the dosage and discontinuing steroids in patients with steroid-dependent UC. PMID:28924114

Long-interval Cytapheresis as a Novel Therapeutic Strategy Leading to Dosage Reduction and Discontinuation of Steroids in Steroid-dependent Ulcerative Colitis.

PubMed

Iizuka, Masahiro; Etou, Takeshi; Kumagai, Makoto; Matsuoka, Atsushi; Numata, Yuka; Sagara, Shiho

2017-10-15

Objective This study was performed to confirm the efficacy of long-interval cytapheresis on steroid-dependent ulcerative colitis (UC). Methods To discontinue steroids in patients with steroid-dependent UC, we previously designed a novel regimen of cytapheresis (CAP), which we termed "long-interval cytapheresis (LI-CAP)", in which CAP was performed as one session every two or three weeks and continued during the whole period of tapering steroid dosage. In this study, we performed LI-CAP therapy 20 times (11 male and 9 female; mean age 41.8 years) between April 2010 and April 2015 for 14 patients with steroid-dependent UC. We evaluated the effectiveness of LI-CAP by examining the improvement in Lichtiger's clinical activity index (CAI), the rate of clinical remission, and the rate of steroid discontinuation. We further examined the rate of sustained steroid-free clinical remission at 6 and 12 months after LI-CAP in patients who successfully discontinued steroid-use after LI-CAP. The primary endpoint was the rate of discontinuation of steroids after LI-CAP. Results The mean CAI score before LI-CAP (7.550) significantly decreased to 1.65 after LI-CAP (p<0.0001). The rate of clinical remission after LI-CAP was 80%. The rate of steroid discontinuation after LI-CAP was 60.0%. The mean dose of daily prednisolone was significantly decreased after LI-CAP (2.30 mg) compared with that before therapy (17.30 mg) (p=0.0003). The rate of sustained steroid-free clinical remission after LI-CAP was 66.7% at 6 months and 66.7% at 12 months. Conclusion We confirmed that LI-CAP has therapeutic effects on reducing the dosage and discontinuing steroids in patients with steroid-dependent UC.

Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins

PubMed Central

Gimber, Niclas; Tadeus, Georgi; Maritzen, Tanja; Schmoranzer, Jan; Haucke, Volker

2015-01-01

Neurotransmission relies on the calcium-triggered exocytic fusion of non-peptide neurotransmitter-containing small synaptic vesicles (SVs) with the presynaptic membrane at active zones (AZs) followed by compensatory endocytic retrieval of SV membranes. Here, we study the diffusional fate of newly exocytosed SV proteins in hippocampal neurons by high-resolution time-lapse imaging. Newly exocytosed SV proteins rapidly disperse within the first seconds post fusion until confined within the presynaptic bouton. Rapid diffusional spread and confinement is followed by slow reclustering of SV proteins at the periactive endocytic zone. Confinement within the presynaptic bouton is mediated in part by SV protein association with the clathrin-based endocytic machinery to limit diffusional spread of newly exocytosed SV proteins. These data suggest that diffusion, and axonal escape of newly exocytosed vesicle proteins, are counteracted by the clathrin-based endocytic machinery together with a presynaptic diffusion barrier. PMID:26399746

Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins

NASA Astrophysics Data System (ADS)

Gimber, Niclas; Tadeus, Georgi; Maritzen, Tanja; Schmoranzer, Jan; Haucke, Volker

2015-09-01

Neurotransmission relies on the calcium-triggered exocytic fusion of non-peptide neurotransmitter-containing small synaptic vesicles (SVs) with the presynaptic membrane at active zones (AZs) followed by compensatory endocytic retrieval of SV membranes. Here, we study the diffusional fate of newly exocytosed SV proteins in hippocampal neurons by high-resolution time-lapse imaging. Newly exocytosed SV proteins rapidly disperse within the first seconds post fusion until confined within the presynaptic bouton. Rapid diffusional spread and confinement is followed by slow reclustering of SV proteins at the periactive endocytic zone. Confinement within the presynaptic bouton is mediated in part by SV protein association with the clathrin-based endocytic machinery to limit diffusional spread of newly exocytosed SV proteins. These data suggest that diffusion, and axonal escape of newly exocytosed vesicle proteins, are counteracted by the clathrin-based endocytic machinery together with a presynaptic diffusion barrier.

Muscle-enhancing behaviors among adolescent girls and boys.

PubMed

Eisenberg, Marla E; Wall, Melanie; Neumark-Sztainer, Dianne

2012-12-01

Media images of men and women have become increasingly muscular, and muscle-enhancing techniques are available to youth. Identifying populations at risk for unhealthy muscle-enhancingbehaviors is of considerable public health importance. The current study uses a large and diverse population-based sample of adolescents to examine the prevalence of muscle-enhancing behaviors and differences across demographic characteristics, weight status, and sports team involvement. Survey data from 2793 diverse adolescents (mean age = 14.4) were collected at 20 urban middle and high schools. Use of 5 muscle-enhancing behaviors was assessed (changing eating, exercising, protein powders, steroids and other muscle-enhancing substances), and a summary score reflecting use of 3 or more behaviors was created. Logistic regression was used to test for differences in each behavior across age group, race/ethnicity, socioeconomic status, BMI category, and sports team participation. Muscle-enhancing behaviors were common in this sample for both boys and girls. For example, 34.7% used protein powders or shakes and 5.9% reported steroid use. Most behaviors were significantly more common among boys. In models mutually adjusted for all covariates, grade level, Asian race, BMI category, and sports team participation were significantly associated with the use of muscle-enhancing behaviors. For example, overweight (odds ratio = 1.45) and obese (odds ratio = 1.90) girls had significantly greater odds of using protein powders or shakes than girls of average BMI. The use of muscle-enhancing behaviors is substantially higher than has been previously reported and is cause for concern. Pediatricians and other health care providers should ask their adolescent patients about muscle-enhancing behaviors.

A molecular and isotopic study of the organic matter from the Paris Basin, France

NASA Technical Reports Server (NTRS)

Lichtfouse, E.; Albrecht, P.; Behar, F.; Hayes, J. M.

1994-01-01

Thirteen Liassic sedimentary rocks of increasing depth and three petroleums from the Paris Basin were studied for 13C/12C isotopic compositions and biological markers, including steranes, sterenes, methylphenanthrenes, methylanthracenes, and triaromatic steroids. The isotopic compositions of n-alkanes from mature sedimentary rocks and petroleums fall in a narrow range (2%), except for the deepest Hettangian rock and the Trias petroleum, for which the short-chain n-alkanes are enriched and depleted in 13C, respectively. Most of the molecular parameters increase over the 2000-2500 m depth range, reflecting the transformation of the organic matter at the onset of petroleum generation. In this zone, carbonate content and carbon isotopic composition of carbonates, as well as molecular parameters, are distinct for the Toarcian and Hettangian source rocks and suggest a migration of organic matter from these two formations. Two novel molecular parameters were defined for this task: one using methyltriaromatic steroids from organic extracts; the other using 1-methylphenanthrene and 2-methylanthracene from kerogen pyrolysates. The anomalous high maturity of the Dogger petroleum relative to the maturity-depth trend of the source rocks is used to estimate the minimal vertical distance of migration of the organic matter from the source rock to the reservoir.

Steroid-antivirals treatment versus steroids alone for the treatment of Bell's palsy: a meta-analysis.

PubMed

Dong, Yabing; Zhu, Yong; Ma, Chuan; Zhao, Huaqiang

2015-01-01

To illustrate whether the steroid-antivirals treatment could acquire a better recovery in patients with Bell's palsy than the steroids alone treatment. We conducted an exhaustive search over Pub med/Medline, Ovid, Elsevier search engines and the Cochrane library thereby collecting the randomized controlled trials in the treatment of patients with Bell's palsy with steroid-antivirals and steroids. The qualities of relevant articles were assessed by GRADE, which was used to present the overall quality of evidence as recommended by the Cochrane Handbook for Systematic Reviews of Interventions. Two investigators evaluated these papers independently, and resolved the disagreements by discussion. At last 8 eligible papers (1816 patients included: 896 treated with steroid-antivirals and 920 treated with steroids alone) match the criteria. Owing to the result (chi(2) = 12.57, P = 0.08, I(2) = 44%) presented by the formal test for heterogeneity, the fixed effect meta-analysis model was chosen. The facial muscle recovery between the steroids-antivirals group and the steroids alone group show significant differences (OR = 1.52, 95% CI: 1.20-1.94), while the statistical outcome of adverse effect shows no statistical significance (OR = 1.28, 95% CI: 0.71-2.31). The present meta-analysis indicates that the steroid-antivirals treatment could improve the recovery rate in patients with Bell's palsy when comparing with the steroid alone treatment. This meta-analysis showed that the steroid-antivirals treatment achieved the better outcomes in patients with Bell's palsy. Clinicians should consider that steroid-antivirals therapy is an alternative choice for the patients with Bell's palsy.

The Carcinogenic Potential of JP-8 and Tungsten in C57BL/6 Mice

DTIC Science & Technology

2011-01-31

acids ; steroid hormone production PHF20, PHD finger protein 20; possible transcription factor ↓PPAP2A, phosphatidic acid phosphatase...and osmY stress promoter-genes and inhibition of enzymes with nucleic- acid substrates [5]. Recent research has demonstrated variable degrees of in...type 2A; dephosphorylating lysophosphatidic acid (LPA) in platelets which terminates signaling actions of LPA. PPARG, ↓RAD23A, RAD23 homolog

Analysis of the Glucocorticoid Receptor in Spontaneous and Drug-induced Steroid Resistant Mutants Isolated from the Human Leukemic Cell Line CEM-C7

DTIC Science & Technology

1991-01-04

be due to defects in GR function. However, the loss of GR function in x" S49 cells is paralleled by the loss of GR mRNA which is not seen in x’ OEM ...in the protein transcription factor IIIA from Xepopus oocytes. EMBO J. 4: 1609-1614. Milgrom. E.; Atger, M.; Baulieu, E.-E. 1973 Acidophilic

A high-throughput UPC2-MS/MS method for the separation and quantification of C19 and C21 steroids and their C11-oxy steroid metabolites in the classical, alternative, backdoor and 11OHA4 steroid pathways.

PubMed

du Toit, Therina; Stander, Maria A; Swart, Amanda C

2018-03-30

In the present study an ultra-performance convergence chromatography tandem mass spectrometry (UPC 2 -MS/MS) analytical method was developed and validated for the determination of 17 C 19 and 14 C 21 steroids, including C11-oxy C 19 and C11-oxy C 21 steroids. The limit of detection and limit of quantification ranged from 0.01 to 10 ng/mL and from 0.01 to 20 ng/mL, respectively, and the method shows the recovery, matrix effect and process efficiency of steroids isolated from a serum matrix to be within acceptable limits. Good accuracy, repeatability and reproducibility were also shown and the method provided excellent sensitivity and selectivity as stereoisomers and regioisomers were also resolved and quantified accurately. Clinical conditions such as congenital adrenal hyperplasia, polycystic ovary syndrome in females and disorders of sex development in neonates and in children, amongst others, are characterized by abnormal steroid levels. Steroid profiling is essential to accurately diagnose steroid levels in the above settings as well as in androgen excess or deficiency in adrenal-linked endocrine diseases. Our method, separating C 19 and C 21 steroids in a single chromatographic step, offers a reduced sample turnover rate in the clinical setting, while providing comprehensive steroid profiles of in vivo steroids in the nmol/L range. This is, to our knowledge, the first method reported to simultaneously separate C 19 and C 21 steroids, together with their C11-hydroxy and C11-keto metabolites -one which may hold promise in the identification of new steroid markers in steroid-linked endocrine diseases, in addition to profiling steroid metabolism and abnormal enzyme activity in patients. Copyright © 2018 Elsevier B.V. All rights reserved.

The effect of real and simulated time-zone shifts upon the circadian rhythms of body temperature, plasma 11-hydroxycorticosteroids, and renal excretion in human subjects

PubMed Central

Elliott, Ann L.; Mills, J. N.; Minors, D. S.; Waterhouse, J. M.

1972-01-01

1. Observations were made upon five subjects who flew through 4½-6 time zones, four of them returning later to their starting point, and upon twenty-three subjects experiencing simulated 6 or 8 hr time zones shifts in either direction in an isolation unit. 2. Measurements were made of plasma concentration of 11-hydroxycorticosteroids, of body temperature, and of urinary excretion of sodium, potassium and chloride. Their rhythm was defined, where possible, by fitting a sine curve of period 24 hr to each separate 24-hr stretch of data and computing the acrophase, or maximum predicted by the sine curve. 3. The adaptation of the plasma steroid rhythm was assessed by the presence of a sharp fall in concentration after the sample collected around 08.00 hr. The time course of adaptation varied widely between individuals; it was usually largely complete by the fourth day after westward, and rather later after eastward, flights. After time shift the pattern often corresponded neither to an adapted nor to an unadapted one, and in a subject followed for many months after a real flight a normal amplitude only appeared 2-3 months after flight. 4. Temperature rhythm adapted by a movement of the acrophase, without change in amplitude, although on some days no rhythm could be observed. This movement was always substantial even on the first day, and was usually nearly complete by the fifth. 5. High nocturnal excretion of electrolyte was often seen in the early days after time shift, more notably after simulated westward flights. Adaptation of urinary electrolyte rhythms usually proceeded as with temperature, but the movement of the acrophase was slower, more variable between individuals, more erratic, and sometimes reversed after partial adaptation. On a few days there were two maxima corresponding to those expected on real and on experimental time. 6. Sodium excretion was much less regular than that of potassium, but adapted more rapidly to time shift, so that the two often became completely dissociated. Chloride behaved much as sodium. 7. The time course of adaptation of the plasma steroid and urinary potassium rhythms were sufficiently similar to suggest a causal connexion. The time course of adaptation of the temperature rhythm did not coincide with that of any other component considered here. PMID:5016984

Free-Flow Zone Electrophoresis of Peptides and Proteins in PDMS Microchip for Narrow pI Range Sample Prefractionation Coupled with Mass Spectrometry

PubMed Central

Song, Yong-Ak; Chan, Michael; Celio, Chris; Tannenbaum, Steven R.; Wishnok, John S.; Han, Jongyoon

2010-01-01

In this paper, we are evaluating the strategy of sorting peptides / proteins based on the charge to mass without resorting to ampholytes and / or isoelectric focusing, using a single- and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems, and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration, and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS. PMID:20163146

21 CFR 1308.26 - Excluded veterinary anabolic steroid implant products.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 9 2013-04-01 2013-04-01 false Excluded veterinary anabolic steroid implant... SCHEDULES OF CONTROLLED SUBSTANCES Excluded Veterinary Anabolic Steroid Implant Products § 1308.26 Excluded veterinary anabolic steroid implant products. (a) Products containing an anabolic steroid, that are expressly...

21 CFR 1308.26 - Excluded veterinary anabolic steroid implant products.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 9 2014-04-01 2014-04-01 false Excluded veterinary anabolic steroid implant... SCHEDULES OF CONTROLLED SUBSTANCES Excluded Veterinary Anabolic Steroid Implant Products § 1308.26 Excluded veterinary anabolic steroid implant products. (a) Products containing an anabolic steroid, that are expressly...

21 CFR 1308.26 - Excluded veterinary anabolic steroid implant products.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 9 2012-04-01 2012-04-01 false Excluded veterinary anabolic steroid implant... SCHEDULES OF CONTROLLED SUBSTANCES Excluded Veterinary Anabolic Steroid Implant Products § 1308.26 Excluded veterinary anabolic steroid implant products. (a) Products containing an anabolic steroid, that are expressly...

21 CFR 1308.26 - Excluded veterinary anabolic steroid implant products.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Excluded veterinary anabolic steroid implant... SCHEDULES OF CONTROLLED SUBSTANCES Excluded Veterinary Anabolic Steroid Implant Products § 1308.26 Excluded veterinary anabolic steroid implant products. (a) Products containing an anabolic steroid, that are expressly...

21 CFR 1308.26 - Excluded veterinary anabolic steroid implant products.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 9 2011-04-01 2011-04-01 false Excluded veterinary anabolic steroid implant... SCHEDULES OF CONTROLLED SUBSTANCES Excluded Veterinary Anabolic Steroid Implant Products § 1308.26 Excluded veterinary anabolic steroid implant products. (a) Products containing an anabolic steroid, that are expressly...

Plasma sex-steroid binding protein in a seasonally breeding reptile, Alligator mississippiensis.

PubMed

Ho, S M; Lance, V; Megaloudis, M

1987-01-01

The properties of a sex-steroid binding protein (SSBP) in the plasma of the American alligator, Alligator mississippiensis, were partially characterized. Alligator SSBP has a sedimentation coefficient of 4S in a 5-20% sucrose gradient. It binds to estradiol-17 beta (E2) and testosterone (T) with limited capacities and moderate affinities (association constant for [3H]E2 is 4.70 +/- 0.09 X 10(8) M-1 and for [3H]T is 1.05 +/- 0.07 X 10(8) M-1, mean +/- SEM of six determinations). Plasma SSBP level, as measured by plasma [3H]E2 binding capacity, varies from 30 to 140 nmol per liter plasma (nM) and was found to be dependent on the gender, sexual maturity, and reproductive state of the animal. Distinct annual fluctuations in plasma SSBP level were observed in female alligators. In adult females, plasma SSBP levels were high (122 +/- 6 nM) in the fall during the nonbreeding season and low (30-60 nM) in spring and early summer during the breeding season. A minimum (33 +/- 6 nM) was reached in mid-June coinciding with the time of oviposition and rapid decline in circulating estrogen levels. This decline in adult female plasma SSBP levels during the breeding season was not observed in immature females. On the contrary, plasma SSBP levels in immature females increased from 81 +/- 14 nM in April to 134 +/- 9 nM in June. Plasma SSBP levels in male alligators showed little changes throughout the entire breeding season; they remained within the range of 80-100 nM from March to June. We believe that seasonal fluctuations in plasma SSBP levels constitute part of the mechanism involved in the regulation of free steroid delivered to target organs in female alligators and that such a mechanism does not exist in male animals.

Obesity during pregnancy affects sex steroid concentrations depending on fetal gender.

PubMed

Maliqueo, M; Cruz, G; Espina, C; Contreras, I; García, M; Echiburú, B; Crisosto, N

2017-11-01

It is not clear whether maternal obesity along with fetal gender affect sex steroid metabolism during pregnancy. Therefore, we compared sex steroid concentrations and placental expression of steroidogenic enzymes between non-obese and obese pregnant women with non-pathological pregnancies, and investigated the influence of fetal gender on these parameters. In 35 normal weight (body mass index (BMI) 20-24.9 kg m - 2 ) (controls) and 36 obese women (BMI 30-36 kg m - 2 ) (obese), a fasting blood sample was obtained at first and at third trimester of gestation to measure progesterone, dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, testosterone and estradiol by liquid chromatography-tandem mass spectrometry and estrone by radioimmunoassay. In a subset of women, placental mRNA and protein expression of steroidogenic enzymes was measured by quantitative PCR and western blot, respectively. The comparisons were primarily made between controls and obese, and then separately according to fetal gender. At first and third trimesters of gestation serum progesterone was lower whereas testosterone was higher in obese women (P<0.05, respectively). Upon analyzing according to fetal gender, lower progesterone levels were present in obese pregnant women with male fetuses at first trimester and with female fetuses at third trimester (P<0.05, respectively). Testosterone was higher in obese women with male fetuses compared to control women with male fetuses (P<0.05). The placental protein expression of P450scc was higher in obese women compared to controls (P<0.05). P450 aromatase was higher in obese women with female fetuses (P=0.009), whereas in obese women with male fetuses P450 aromatase was lower compared to control women (P=0.026). Obesity in non-pathological pregnancies alters the maternal serum progesterone and testosterone concentrations depending on fetal gender. These changes can be attributed to gender-related placental adaptations, as the expression of P450 aromatase is different in placentas from females compared to males.

Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance

PubMed Central

Jensen, Victor L; Li, Chunmei; Bowie, Rachel V; Clarke, Lara; Mohan, Swetha; Blacque, Oliver E; Leroux, Michel R

2015-01-01

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this “ciliary gate” assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS-5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y-shaped axoneme-to-membrane connectors. Coiled-coil and C2 domains within MKS-5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body-associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS-5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP-2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP2 within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment. PMID:26392567

17β-Estradiol Alters Oxidative Stress Response Protein Expression and Oxidative Damage in the Uterus

PubMed Central

Yuan, Lisi; Dietrich, Alicia K.; Nardulli, Ann M.

2014-01-01

The steroid hormone 17β-estradiol (E2) has profound effects on the uterus. However, with the E2-induced increase in uterine cell proliferation and metabolism comes increased production of reactive oxygen species (ROS). We examined the expression of an interactive network of oxidative stress response proteins including thioredoxin (Trx), Cu/Zn superoxide dismutase (SOD1), apurinic endonuclease (Ape1), and protein disulfide isomerase (PDI). We demonstrated that treatment of ovariectomized C57BL/6J female mice with E2 increased the mRNA and protein levels of Trx, but decreased SOD1 and Ape1 mRNA and protein expression. In contrast, E2 treatment increased PDI protein levels but had no effect on PDI transcript levels.Interestingly, E2 treatment also increased two markers of cellular damage, lipid peroxidation and protein carbonylation. Our studies suggest that the decreased expression of SOD1 and Ape1 caused by E2 treatment may in the long term result in disruption of ROS regulation and play a role in endometrial carcinogenesis. PMID:24103313

21 CFR 1308.34 - Exempt anabolic steroid products.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 9 2012-04-01 2012-04-01 false Exempt anabolic steroid products. 1308.34 Section... SUBSTANCES Exempt Anabolic Steroid Products § 1308.34 Exempt anabolic steroid products. The list of compounds, mixtures, or preparations that contain an anabolic steroid that have been exempted by the Administrator...

21 CFR 1308.34 - Exempt anabolic steroid products.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 9 2014-04-01 2014-04-01 false Exempt anabolic steroid products. 1308.34 Section... SUBSTANCES Exempt Anabolic Steroid Products § 1308.34 Exempt anabolic steroid products. The list of compounds, mixtures, or preparations that contain an anabolic steroid that have been exempted by the Administrator...

21 CFR 1308.34 - Exempt anabolic steroid products.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 9 2011-04-01 2011-04-01 false Exempt anabolic steroid products. 1308.34 Section... SUBSTANCES Exempt Anabolic Steroid Products § 1308.34 Exempt anabolic steroid products. The list of compounds, mixtures, or preparations that contain an anabolic steroid that have been exempted by the Administrator...

21 CFR 1308.34 - Exempt anabolic steroid products.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 9 2013-04-01 2013-04-01 false Exempt anabolic steroid products. 1308.34 Section... SUBSTANCES Exempt Anabolic Steroid Products § 1308.34 Exempt anabolic steroid products. The list of compounds, mixtures, or preparations that contain an anabolic steroid that have been exempted by the Administrator...

Steroids in Athletics: One University's Experience.

ERIC Educational Resources Information Center

Lopez, Mike

1990-01-01

Presents an account of one university's experience in conducting an investigation into possible steroid use by student athletes and the development of a program to deal with the problem. Discusses why athletes use steroids and how steroids are taken. Concludes it is likely many steroid-related deaths of athletes go undetected. (Author/ABL)

21 CFR 1308.34 - Exempt anabolic steroid products.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Exempt anabolic steroid products. 1308.34 Section... SUBSTANCES Exempt Anabolic Steroid Products § 1308.34 Exempt anabolic steroid products. The list of compounds, mixtures, or preparations that contain an anabolic steroid that have been exempted by the Administrator...

Adolescent Steroid Use.

ERIC Educational Resources Information Center

Office of Inspector General (DHHS), Washington, DC.

The study focused on non-medical steroid use by adolescents according to data obtained from the National Institute on Drug Abuse, professional literature, 30 key informants knowledgeable in steroid issues, and 72 current or former steroid users. The findings indicated: (1) over 250,000 adolescents, primarily males, used or have used steroids, and…

Quest for steroidomimetics: Amino acids derived steroidal and nonsteroidal architectures.

PubMed

Shagufta; Ahmad, Irshad; Panda, Gautam

2017-06-16

The chiral pool amino acids have been utilized for the construction of steroidal and non-steroidal architectures in the quest for steroidomimetics. Chirality derived from amino acid-based architectures provides new and easy to incorporate chiral chemical space, which is otherwise very difficult to introduce and comprised of several synthetic steps for asymmetric steroids. The different and exciting ligand-receptor interactions may arise from the use of each amino acid enantiomer that was introduced into the chiral steroidal backbone. The A and D rings of steroidal architectures can be mimicked by the phenyl group of the amino acid tyrosine. The Mitsunobu reaction, nucleophilic substitution and elimination, etc. were utilized for constructing diverse tri- and tetracyclic steroidal skeletons as well as benzofused seco-steroids from amino acids. These benzofused, amino acid-derived steroidal and nonsteroidal molecules had promising biological activity in hormonal related disorders. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Characterizing the distribution of steroid sulfatase during embryonic development: when and where might metabolites of maternal steroids be reactivated?

PubMed

Paitz, Ryan T; Duffield, Kristin R; Bowden, Rachel M

2017-12-15

All vertebrate embryos are exposed to maternally derived steroids during development. In placental vertebrates, metabolism of maternal steroids by the placenta modulates embryonic exposure, but how exposure is regulated in oviparous vertebrates is less clear. Recent work in oviparous vertebrates has demonstrated that steroids are not static molecules, as they can be converted to more polar steroid sulfates by sulfotransferase enzymes. Importantly, these steroid sulfates can be converted back to the parent compound by the enzyme steroid sulfatase (STS). We investigated when and where STS was present during embryonic development in the red-eared slider turtle, Trachemys scripta We report that STS is present during all stages of development and in all tissues we examined. We conclude that STS activity may be particularly important for regulating maternal steroid exposure in oviparous vertebrates. © 2017. Published by The Company of Biologists Ltd.

Effects of Sex Steroids in the Human Brain.

PubMed

Nguyen, Tuong-Vi; Ducharme, Simon; Karama, Sherif

2017-11-01

Sex steroids are thought to play a critical developmental role in shaping both cortical and subcortical structures in the human brain. Periods of profound changes in sex steroids invariably coincide with the onset of sex differences in mental health vulnerability, highlighting the importance of sex steroids in determining sexual differentiation of the brain. Yet, most of the evidence for the central effects of sex steroids relies on non-human studies, as several challenges have limited our understanding of these effects in humans: the lack of systematic assessment of the human sex steroid metabolome, the different developmental trajectories of specific sex steroids, the impact of genetic variation and epigenetic changes, and the plethora of interactions between sex steroids, sex chromosomes, neurotransmitters, and other hormonal systems. Here we review how multimodal strategies may be employed to bridge the gap between the basic and clinical understanding of sex steroid-related changes in the human brain.

Effect of oxidizing adulterants on human urinary steroid profiles.

PubMed

Kuzhiumparambil, Unnikrishnan; Fu, Shanlin

2013-02-01

Steroid profiling is the most versatile and informative technique adapted by doping control laboratories for detection of steroid abuse. The absolute concentrations and ratios of endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol constitute the significant characteristics of a steroid profile. In the present study we report the influence of various oxidizing adulterants on the steroid profile of human urine. Gas chromatography-mass spectrometry analysis was carried out to develop the steroid profile of human male and female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate, cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium dichromate and potassium perchlorate were reacted with urine at various concentrations and conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the oxidizing chemicals led to significant changes in endogenous steroid profile parameters which were considered stable under normal conditions. These oxidizing chemicals can cause serious problems regarding the interpretation of steroid profiles and have the potential to act as masking agents that can complicate or prevent the detection of the steroid abuse. Copyright © 2012 Elsevier Inc. All rights reserved.

Development and application of a high-throughput sample cleanup process based on 96-well plate for simultaneous determination of 16 steroids in biological matrices using liquid chromatography-triple quadrupole mass spectrometry.

PubMed

Luo, Guanzhong; Li, Youxin; Bao, James J

2016-02-01

A novel high-throughput sample pretreatment system was developed by the integration of protein precipitation (PP), phospholipid removal (PPR), and hollow fiber liquid-phase microextraction (HF-LPME) into two simple 96-well plates and a matching 96-grid lid. With this system, 16 steroids were separated from biological matrices of plasma, milk, and urine and analyzed by liquid chromatography-triple quadrupole mass spectrometry. In the tandem sample cleanup process, the prepositive PP and PPR step preliminarily removed some of the interferences from the biological matrices. The following HF-LPME step kept the residual interference out of the hollow fiber and enriched the steroids in the hollow fiber to achieve high sensitivity. By a series of method optimizations, acetonitrile was chosen as the crash solvent for PP and PPR. A mixture of octanol and toluene (1:1 v/v) was used as the acceptor phase for HF-LPME. The extraction was conducted at 80 rpm for 50 min in a donor phase containing 1 mL 20% sodium chloride at 25 °C. Under these conditions, the limits of detection for the 16 steroids were 3.6-300.0 pg(.)mL(-1) in plasma, 3.0-270.0 pg·mL(-1) in milk, and 2.2-210.0 pg(.)mL(-1) in urine. The recoveries of the 16 steroids were 81.9-97.9% in plasma (relative standard deviation 1.0-8.0%), 80.6-97.7% in milk (relative standard deviation 0.8-5.4%), and 87.3-98.7% in urine (relative standard deviation 1.0-4.9%). Further, the integrated 96-well platform of PP, PPR, and HF-LPME enabled us to run this assay in an automatic and high-throughput fashion. The reliability of the method was further corroborated by evaluation of its applicability in plasma and urine samples from volunteers and fresh bovine milk from local dairy enterprises.

Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster.

PubMed

Tolhuis, Bas; de Wit, Elzo; Muijrers, Inhua; Teunissen, Hans; Talhout, Wendy; van Steensel, Bas; van Lohuizen, Maarten

2006-06-01

Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila melanogaster Kc cells. We found that Pc associates with large genomic regions of up to approximately 150 kb in size, hereafter referred to as 'Pc domains'. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 Lys27 and is generally transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains.

Simultaneous quantification of five steroid saponins from Dioscorea zingiberensis C.H.Wright in rat plasma by HPLC-MS/MS and its application to the pharmacokinetic studies

PubMed Central

Zhang, Xinxin; Li, Jing; Ito, Yoichiro; Sun, Wenji

2014-01-01

A simple, reliable and sensitive high-performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) was established for simultaneous analyses of the following 5 steroid saponins in rat plasma after the single dose administration of total steroid saponins extracted from the rhizome of Dioscorea zingiberensis C.H.Wright for the first time. Protodioscin, huangjiangsu A, zingiberensis new saponin, dioscin, and gracillin were quantified using ginsenoside Rb1 as the internal standard (IS). The plasma samples were pretreated by a single step acetonitrile-mediated protein precipitation. The chromatographic separation was performed on an Inersil ODS-3 C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase composed of acetonitrile and water containing 0.1% formic acid under a gradient elution mode at 0.2 mL min−1 using a microsplit after the eluent from the HPLC apparatus. The quantification was accomplished on a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) in the positive ionization mode. The above five analytes were stable under sample storage and preparation conditions applied in the present study. The linearity, precision, accuracy, and recoveries of the analysis confirmed the requirements for quality-control purposes. After validation, this proposed method was successfully adopted to investigate the pharmacokinetic parameters of these five analytes. PMID:25201262

Prostaglandins, steroids and reception (an attempt to model the structure of the active centers of adrenoreception).

PubMed

Podymov, V K; Piruzyan, L A; Gladkikh, S P; Kats, M M; Nizhnii, S V

1980-01-01

On the basis of numerous results of investigations on adrenergic systems, an orientational model of the adrenoreceptor (AR) is postulated. Its active center includes low-molecular-weight components--prostaglandins (PGE, PGF), steroids (cortisone, hydrocortisone), S+-adenosylmethionine, Ca, Mg, and Mn ions. Appraisal of the stereospecific characteristics of such a functional unit of AR explains the difference in the nature and magnitude of the effects of interaction of the catecholamines, their agonists and antagonists will the so-called alpha- and beta-AR. Depending on the organ or tissue in which the AR is located, its protein subunits comprise adenylcyclase (beta-AR) or Na,K-ATPase (alpha-AR). An obligatory component of the AR is catechol-O-methyltransferase. The model elaborated describes satisfactorily the molecular mechanisms of action of many pharmacological agents, explains why attempts to isolate and reconstruct the AR have proved fruitless, and gives grounds for rejecting the hypothesis that there exist steroid, prostaglandin, and purinergic receptors, linking the exceptionally high and diverse activity of these biologically active substances with their participation in adrenoreception among other reasons. A conception of the active centers of the AR as low-molecular-weight entities permits the explanation of such phenomena as the desensitization of the AR, the "interconversion" of beta-AR into alpha-AR with a change in the parameters of the medium, and certain components of the pathogenesis of bronchial asthma, etc.

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

PubMed Central

Xu, Li-Qin; Liu, Yong-Jun; Yao, Kang; Liu, Hao-Hao; Tao, Xin-Yi; Wang, Feng-Qing; Wei, Dong-Zhi

2016-01-01

The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively. PMID:26898409

Anabolic steroid and gender-dependent modulation of cytosolic HSP70s in fast- and slow-twitch skeletal muscle.

PubMed

González, B; Hernando, R; Manso, R

2000-09-01

Besides their clinical uses, anabolic steroids (AASs) are self-administered by athletes to improve muscle mass and sports performance. The biological basis for their presumed effectiveness at suprapharmacological doses, however, remains uncertain. Since the expression of high levels of some stress proteins (HSPs) has been associated with an increased tolerance to stress and chronic exercise up-regulates HSP72 in skeletal muscle, this investigation was aimed at testing whether the administration of suprapharmacological doses of AASs, either alone or in conjunction with chronic exercise, induced changes in HSP72. Nandrolone decanoate (ND), an estrene derivative, but not stanozolol (ST), a derivative of the androstane series, up-regulated the levels of HSP72 and changed the proportions of various charge variants of the cytosolic HSP70s in sedentary and exercise-trained rats, exclusively in fast-twitch fibres. Since the expression of HSP73-levels in skeletal muscle was dependent on gender but not on muscle type, and that of HSP72-levels was muscle type specific but gender-independent, ND effects on cytosolic HSP70s could not be explained solely by a functional relationship with sex steroids. The reported results indicate that, by up-regulating the expression levels of HSP72 in fast-twitch fibres, nandrolone decanoate could contribute to improving the tolerance of skeletal muscle to high-intensity training.

Cyclosporine treatment of steroid-refractory ulcerative colitis during pregnancy.

PubMed

Branche, Julien; Cortot, Antoine; Bourreille, Arnaud; Coffin, Benoît; de Vos, Martine; de Saussure, Philippe; Seksik, Philippe; Marteau, Philippe; Lemann, Marc; Colombel, Jean-Frédéric

2009-07-01

Cyclosporine is considered a safe and effective treatment of severe steroid-refractory ulcerative colitis (UC). However, few data are available concerning its safety profile in pregnant women. We report here the experience of 5 GETAID centers. In a retrospective study data on patients with severe UC treated with cyclosporine during pregnancy were extracted from medical records of consecutive patients treated between 2001 and 2007. Eight patients (median age 30.5 years old) were identified. At the time of flare-up the median duration of pregnancy was 11.5 weeks of gestation (range 4-25). Seven patients had pancolitis. All patients had more than 3 commonly used clinical and biological severity criteria. Three patients had severe endoscopic lesions and 5 patients had not. All patients received intravenous corticosteroids for at least 7 days before introduction of cyclosporine. Two patients received azathioprine during treatment with cyclosporine. No severe infections or other complications due to treatment were observed. Treatment was effective in 7/8 patients. One patient received infliximab due to cyclosporine therapy failure with a good outcome. No colectomy was performed during pregnancy. Seven pregnancies were conducted to term, but 1 in utero death occurred due to maternal absence of S-protein. Two newborns were premature, including 1 case of hypotrophy. No malformations were observed. In our experience, treatment with cyclosporine for steroid-refractory UC during pregnancy can be considered safe and effective.

Downregulation of peroxisome proliferator-activated receptors (PPARs) in nasal polyposis

PubMed Central

Cardell, Lars-Olaf; Hägge, Magnus; Uddman, Rolf; Adner, Mikael

2005-01-01

Background Peroxisome proliferator-activated receptor (PPAR) α, βδ and γ are nuclear receptors activated by fatty acid metabolites. An anti-inflammatory role for these receptors in airway inflammation has been suggested. Methods Nasal biopsies were obtained from 10 healthy volunteers and 10 patients with symptomatic allergic rhinitis. Nasal polyps were obtained from 22 patients, before and after 4 weeks of local steroid treatment (fluticasone). Real-time RT-PCR was used for mRNA quantification and immunohistochemistry for protein localization and quantification. Results mRNA expression of PPARα, PPARβδ, PPARγ was found in all specimens. No differences in the expression of PPARs were obtained in nasal biopsies from patients with allergic rhinitis and healthy volunteers. Nasal polyps exhibited lower levels of PPARα and PPARγ than normal nasal mucosa and these levels were, for PPARγ, further reduced following steroid treatment. PPARγ immunoreactivity was detected in the epithelium, but also found in smooth muscle of blood vessels, glandular acini and inflammatory cells. Quantitative evaluation of the epithelial immunostaining revealed no differences between nasal biopsies from patients with allergic rhinitis and healthy volunteers. In polyps, the PPARγ immunoreactivity was lower than in nasal mucosa and further decreased after steroid treatment. Conclusion The down-regulation of PPARγ, in nasal polyposis but not in turbinates during symptomatic seasonal rhinitis, suggests that PPARγ might be of importance in long standing inflammations. PMID:16271155

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism.

PubMed

Xu, Li-Qin; Liu, Yong-Jun; Yao, Kang; Liu, Hao-Hao; Tao, Xin-Yi; Wang, Feng-Qing; Wei, Dong-Zhi

2016-02-22

The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.

The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.

PubMed

Ewen, Kerstin M; Schiffler, Burkhard; Uhlmann-Schiffler, Heike; Bernhardt, Rita; Hannemann, Frank

2008-05-01

Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

The place of hyperbaric oxygen therapy and ozone therapy in sudden hearing loss.

PubMed

Ergun Taşdöven, Gülin; Derin, Alper Tunga; Yaprak, Neslihan; Özçağlar, Hasan Ümit

It is difficult to evaluate the effect of drugs clinically used for idiopathic sudden sensorineural hearing loss, mainly because its underlying mechanism remains unknown. This study assessed the efficacy of hyperbaric oxygen therapy or ozone therapy in the treatment of idiopathic sudden sensorineural hearing loss, when either therapy was included with steroid treatment. A retrospective analysis examined 106 patients with idiopathic sudden sensorineural hearing loss seen between January 2010 and June 2012. Those with an identified etiology were excluded. The patients were divided into three treatment groups: oral steroid only (n=65), oral steroid+hyperbaric oxygen (n=26), and oral steroid+ozone (n=17). Treatment success was assessed using Siegel criteria and mean gains using pre- and post-treatment audiograms. The highest response rate to treatment was observed in the oral steroid+ozone therapy group (82.4%), followed by the oral steroid+hyperbaric oxygen (61.5%), and oral steroid groups (50.8%). There were no significant differences in the response to treatment between the oral steroid and oral steroid+hyperbaric oxygen groups (p<0.355). The oral steroid+ozone group showed a significantly higher response rate to treatment than the oral steroid group (p=0.019). There were no significant differences between the oral steroid+hyperbaric oxygen and oral steroid+ozone groups (p=0.146). The efficiency of steroid treatment in patients with severe hearing loss was low. It was statistically ascertained that adding hyperbaric oxygen or ozone therapy to the treatment contributed significantly to treatment success. Copyright © 2016 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

Steroid-antivirals treatment versus steroids alone for the treatment of Bell’s palsy: a meta-analysis

PubMed Central

Dong, Yabing; Zhu, Yong; Ma, Chuan; Zhao, Huaqiang

2015-01-01

Background: To illustrate whether the steroid-antivirals treatment could acquire a better recovery in patients with Bell’s palsy than the steroids alone treatment. Materials and methods: We conducted an exhaustive search over Pub med/Medline, Ovid, Elsevier search engines and the Cochrane library thereby collecting the randomized controlled trials in the treatment of patients with Bell’s palsy with steroid-antivirals and steroids. The qualities of relevant articles were assessed by GRADE, which was used to present the overall quality of evidence as recommended by the Cochrane Handbook for Systematic Reviews of Interventions. Results: Two investigators evaluated these papers independently, and resolved the disagreements by discussion. At last 8 eligible papers (1816 patients included: 896 treated with steroid-antivirals and 920 treated with steroids alone) match the criteria. Owing to the result (chi2 = 12.57, P = 0.08, I2 = 44%) presented by the formal test for heterogeneity, the fixed effect meta-analysis model was chosen. The facial muscle recovery between the steroids-antivirals group and the steroids alone group show significant differences (OR = 1.52, 95% CI: 1.20-1.94), while the statistical outcome of adverse effect shows no statistical significance (OR = 1.28, 95% CI: 0.71-2.31). Conclusions: The present meta-analysis indicates that the steroid-antivirals treatment could improve the recovery rate in patients with Bell’s palsy when comparing with the steroid alone treatment. Clinical significance: This meta-analysis showed that the steroid-antivirals treatment achieved the better outcomes in patients with Bell’s palsy. Clinicians should consider that steroid-antivirals therapy is an alternative choice for the patients with Bell’s palsy. PMID:25785012

Conserved steroid hormone homology converges on NFκB to modulate inflammation in asthma

PubMed Central

Payne, Asha S.; Freishtat, Robert J.

2012-01-01

Asthma is a complex, multifactorial disease comprising multiple different subtypes, rather than a single disease entity [1], yet has a consistent clinical phenotype: recurring episodes of chest tightness, wheezing, and difficulty breathing. Despite the complex pathogenesis of asthma, steroid hormones (e.g. glucocorticoids) are ubiquitous in the acute and chronic management of all types of asthma. Overall, steroid hormones are a class of widely-relevant, biologically-active compounds originating from cholesterol and altered in a stepwise fashion, but maintain a basic 17-carbon, 4-ring structure. Steroids are lipophilic molecules that diffuse readily through cell membranes to directly and/or indirectly affect gene transcription. In addition, they employ rapid, non-genomic actions to affect cellular products. Steroid hormones are comprised of several groups (including glucocorticoids, sex steroid hormones, and secosteroids) with critical divergent biological and physiological functions relevant to health and disease. However, the conserved homology of steroid hormone molecules, receptors, and signaling pathways suggest that each of these is part of dynamic system of hormone interaction, likely involving overlap of downstream signaling mechanisms. Therefore, we will review the similarities and differences of these three groups of steroid hormones (i.e. glucocorticoids, sex steroid hormones, and secosteroids), identifying NFκB as a common inflammatory mediator. Despite our understanding of the impact of individual steroids (e.g. glucocorticoids, sex steroids and secosteroids) on asthma, research has yet to explain the interplay of the dynamic system in which these hormones function. To do so, there needs to be better understanding of the interplay of classical, non-classical, and non-genomic steroid hormone function. However, clues from the conserved homology steroid hormone structure and function and signaling pathways, offer insight into a possible model of steroid hormone regulation of inflammation in asthma through common NFκB-mediated downstream events. PMID:22183120

Quantitative proteomic analysis of age-related subventricular zone proteins associated with neurodegenerative disease.

PubMed

Wang, Xianli; Dong, Chuanming; Sun, Lixin; Zhu, Liang; Sun, Chenxi; Ma, Rongjie; Ning, Ke; Lu, Bing; Zhang, Jinfu; Xu, Jun

2016-11-18

Aging is characterized by a progressive decline in the function of adult tissues which can lead to neurodegenerative disorders. However, little is known about the correlation between protein changes in the subventricular zone (SVZ) and neurodegenerative diseases with age. In the present study, neural stem cells (NSCs) were derived from the SVZ on postnatal 7 d, 1 m, and 12 m-old mice. With age, NSCs exhibited increased SA-β-gal activity and decreased proliferation and pool size in the SVZ zone, and were associated with elevated inflammatory chemokines and cytokines. Furthermore, quantitative proteomics and ingenuity pathway analysis were used to evaluate the significant age-related alterations in proteins and their functions. Some downregulated proteins such as DPYSL2, TPI1, ALDH, and UCHL1 were found to play critical roles in the neurological disease and PSMA1, PSMA3, PSMC2, PSMD11, and UCHL1 in protein homeostasis. Taken together, we have provided valuable insight into the cellular and molecular processes that underlie aging-associated declines in SVZ neurogenesis for the early detection of differences in gene expression and the potential risk of neurological disease, which is beneficial in the prevention of the diseases.

Characteristics of Steroid Users in an Adolescent School Population.

ERIC Educational Resources Information Center

Adlaf, Edward M.; Smart, Reginald G.

1992-01-01

Examined rates of steroid use among Ontario adolescent students. Findings from 3,892 students revealed that 1.1 percent reported using steroids over past year. Steroid users were significantly more likely to use stimulants, caffeine, and relaxants than were nonsteroid users. Demographically, steroid users were significantly more likely to be male…

Steroid avoidance or withdrawal for kidney transplant recipients.

PubMed

Haller, Maria C; Royuela, Ana; Nagler, Evi V; Pascual, Julio; Webster, Angela C

2016-08-22

Steroid-sparing strategies have been attempted in recent decades to avoid morbidity from long-term steroid intake among kidney transplant recipients. Previous systematic reviews of steroid withdrawal after kidney transplantation have shown a significant increase in acute rejection. There are various protocols to withdraw steroids after kidney transplantation and their possible benefits or harms are subject to systematic review. This is an update of a review first published in 2009. To evaluate the benefits and harms of steroid withdrawal or avoidance for kidney transplant recipients. We searched the Cochrane Kidney and Transplant Specialised Register to 15 February 2016 through contact with the Information Specialist using search terms relevant to this review. All randomised and quasi-randomised controlled trials (RCTs) in which steroids were avoided or withdrawn at any time point after kidney transplantation were included. Assessment of risk of bias and data extraction was performed by two authors independently and disagreement resolved by discussion. Statistical analyses were performed using the random-effects model and dichotomous outcomes were reported as relative risk (RR) and continuous outcomes as mean difference (MD) with 95% confidence intervals. We included 48 studies (224 reports) that involved 7803 randomised participants. Of these, three studies were conducted in children (346 participants). The 2009 review included 30 studies (94 reports, 5949 participants). Risk of bias was assessed as low for sequence generation in 19 studies and allocation concealment in 14 studies. Incomplete outcome data were adequately addressed in 22 studies and 37 were free of selective reporting.The 48 included studies evaluated three different comparisons: steroid avoidance or withdrawal compared with steroid maintenance, and steroid avoidance compared with steroid withdrawal. For the adult studies there was no significant difference in patient mortality either in studies comparing steroid withdrawal versus steroid maintenance (10 studies, 1913 participants, death at one year post transplantation: RR 0.68, 95% CI 0.36 to 1.30) or in studies comparing steroid avoidance versus steroid maintenance (10 studies, 1462 participants, death at one year after transplantation: RR 0.96, 95% CI 0.52 to 1.80). Similarly no significant difference in graft loss was found comparing steroid withdrawal versus steroid maintenance (8 studies, 1817 participants, graft loss excluding death with functioning graft at one year after transplantation: RR 1.17, 95% CI 0.72 to 1.92) and comparing steroid avoidance versus steroid maintenance (7 studies, 1211 participants, graft loss excluding death with functioning graft at one year after transplantation: RR 1.09, 95% CI 0.64 to 1.86). The risk of acute rejection significantly increased in patients treated with steroids for less than 14 days after transplantation (7 studies, 835 participants: RR 1.58, 95% CI 1.08 to 2.30) and in patients who were withdrawn from steroids at a later time point after transplantation (10 studies, 1913 participants, RR 1.77, 95% CI 1.20 to 2.61). There was no evidence to suggest a difference in harmful events, such as infection and malignancy, in adult kidney transplant recipients. The effect of steroid withdrawal in children is unclear. This updated review increases the evidence that steroid avoidance and withdrawal after kidney transplantation significantly increase the risk of acute rejection. There was no evidence to suggest a difference in patient mortality or graft loss up to five year after transplantation, but long-term consequences of steroid avoidance and withdrawal remain unclear until today, because prospective long-term studies have not been conducted.

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome.

PubMed

Warejko, Jillian K; Tan, Weizhen; Daga, Ankana; Schapiro, David; Lawson, Jennifer A; Shril, Shirlee; Lovric, Svjetlana; Ashraf, Shazia; Rao, Jia; Hermle, Tobias; Jobst-Schwan, Tilman; Widmeier, Eugen; Majmundar, Amar J; Schneider, Ronen; Gee, Heon Yung; Schmidt, J Magdalena; Vivante, Asaf; van der Ven, Amelie T; Ityel, Hadas; Chen, Jing; Sadowski, Carolin E; Kohl, Stefan; Pabst, Werner L; Nakayama, Makiko; Somers, Michael J G; Rodig, Nancy M; Daouk, Ghaleb; Baum, Michelle; Stein, Deborah R; Ferguson, Michael A; Traum, Avram Z; Soliman, Neveen A; Kari, Jameela A; El Desoky, Sherif; Fathy, Hanan; Zenker, Martin; Bakkaloglu, Sevcan A; Müller, Dominik; Noyan, Aytul; Ozaltin, Fatih; Cadnapaphornchai, Melissa A; Hashmi, Seema; Hopcian, Jeffrey; Kopp, Jeffrey B; Benador, Nadine; Bockenhauer, Detlef; Bogdanovic, Radovan; Stajić, Nataša; Chernin, Gil; Ettenger, Robert; Fehrenbach, Henry; Kemper, Markus; Munarriz, Reyner Loza; Podracka, Ludmila; Büscher, Rainer; Serdaroglu, Erkin; Tasic, Velibor; Mane, Shrikant; Lifton, Richard P; Braun, Daniela A; Hildebrandt, Friedhelm

2018-01-06

Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1 , PLCE1 , NPHS2 , and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome. Copyright © 2018 by the American Society of Nephrology.

Fusion Protein Cytokine Therapy After Rituximab in Treating Patients With B-Cell Non-Hodgkin Lymphoma

ClinicalTrials.gov

2015-06-03

Anaplastic Large Cell Lymphoma; Cutaneous B-cell Non-Hodgkin Lymphoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Intraocular Lymphoma; Nodal Marginal Zone B-cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Marginal Zone Lymphoma; Small Intestine Lymphoma; Splenic Marginal Zone Lymphoma; Testicular Lymphoma; Waldenstrom Macroglobulinemia

Steroids

MedlinePlus

... or sometimes with chemotherapy. Steroids are used for short-term symptom control although they may occasionally be continued ... even in lymphoma steroids are not typically a long-term cure for this tumor. HOW ARE STEROIDS TAKEN? ...

Molecular identification of StAR and 3βHSD1 and characterization in response to GnIH stimulation in protogynous hermaphroditic grouper (Epinephelus coioides).

PubMed

Wang, Qingqing; Qi, Xin; Tang, Haipei; Guo, Yin; Li, Shuisheng; Li, Gaofei; Yang, Xiaoli; Zhang, Haifa; Liu, Xiaochun; Lin, Haoran

2017-04-01

Gonadal steroids are critical factors in reproduction and sex reverse process. StAR (steroidogenic acute regulatory protein), transferring the cholesterol from the outer mitochondrial membrane to the inner membrane, is the rate-limiting factor of steroidogenesis. 3βHSD (3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase), converting Δ5-steroids into Δ4-steroids, is an important oxidoreductase in steroidogenesis. In the present study, StAR and 3βHSD1 were cloned and characterized from protogynous orange-spotted grouper. StAR cDNA contains an 861bp open reading frame (ORF), encoding a predicted protein of 286 amino acids, and the ORF of 3βHSD1 was 1125bp, encoding a predicted protein of 374 amino acids. The transcript of StAR was mainly expressed in gonad, while 3βHSD1 mRNA was predominantly detected in brain and gonad. In the previous study, we found the expression of GnIH mRNA level in male, as well as in 17 alpha-methyltestosterone (MT)-induced male fish was significantly higher than in female fish, this indicating that GnIH/GnIHR signaling might be involved in the regulation of sex reversal and male maintenance. In order to figure out the function of GnIH in steroidogenesis, the expression of StAR and 3βHSD1 regulated by GnIH was examined. In vitro study showed that treatment of cultured ovary fragments with gGnIH peptides significantly stimulated the expression of StAR and 3βHSD1. In addition, the mRNA levels of StAR and 3βHSD1 were significantly increased after intraperitoneal injection (i.p.) with gGnIH peptides. Moreover, during MT-induced sex change from female to male, the levels of StAR mRNA significantly increased by 5.2, 24.8 and 353.5 folds, and that of 3βHSD1 mRNA by 3.5, 32.5 and 55.4 folds at the 2nd, 4th and 6th week after MT implantation, respectively. Collectively, our results indicate that GnIH may be involved in the regulation of sex reversal or male maintenance by stimulating the expression of StAR and 3βHSD1 in protogynous grouper. Copyright © 2017 Elsevier Inc. All rights reserved.

The effect of local use of nandrolone decanoate on rotator cuff repair in rabbits.

PubMed

Papaspiliopoulos, Athanasios; Papaparaskeva, Kleo; Papadopoulou, Eleni; Feroussis, John; Papalois, Apostolos; Zoubos, Aristedes

2010-08-01

There is still controversy about the effect of anabolic steroid on connective tissue. This study examines the hypothesis that the local use of nandrolone decanoate, an anabolic steroid on rotator cuff, facilitates the healing process when used in combination with surgical repair. Forty-eight male rabbits were divided in four groups with anabolic steroids (Nandrolone Decanoate 10 mg/kg) and immobilization as variables. The groups were the following: first group, nonsteroid use-immobilization (NSI); second group, nonsteroid use-nonimmobilization (NSNI); third group, steroid use-immobilization (SI); fourth group steroid use-nonimmobilization (SNI). Every rabbit underwent a rotator cuff incision and reconstruction. Fifteen days later the tendons were sent for biomechanical and histological evaluation. Groups that did not receive anabolic steroids showed better healing and more tendon strength in comparison to groups that received anabolic steroids. Microscopic examination of specimens from the groups without the use of anabolic steroid showed extensive fibroblastic activity whereas the specimens from those groups with anabolic steroid use showed focal fibroblastic reaction and inflammation. Immobilization provided better results in the groups with anabolic steroid use but it did not influence healing in groups without steroids. The effect of local nandrolone decanoate use on a rotator cuff tear is detrimental, acting as a healing inhibitor.

Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

PubMed

Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

2016-02-18

Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease in early xylem zones. We observed upregulation of two forms of xylem cysteine protease (Potri.002G005700.1 and Potri.005G256000.2; Pt-XCP2.1) in early xylem and their downregulation in late maturing xylem. Our data also show that Pt-KOR1.3 (Potri.003G151700.2) exhibits an expression pattern that supports the hypothesis put forward in previous studies that this is a key xyloglucanase involved in cellulose biosynthesis in primary cell walls and reduction of cellulose crystallinity in secondary walls. Our novel multivariate approach highlights important processes and provides confirmatory insights into the molecular foundations of wood development.

Peroxisome proliferator-activated receptor gamma signaling in human sperm physiology

PubMed Central

Liu, Li-Li; Xian, Hua; Cao, Jing-Chen; Zhang, Chong; Zhang, Yong-Hui; Chen, Miao-Miao; Qian, Yi; Jiang, Ming

2015-01-01

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the PPARs, which are transcription factors of the steroid receptor superfamily. PPARγ acts as an important molecule for regulating energy homeostasis, modulates the hypothalamic-pituitary-gonadal (HPG) axis, and is reciprocally regulated by HPG. In the human, PPARγ protein is highly expressed in ejaculated spermatozoa, implying a possible role of PPARγ signaling in regulating sperm energy dissipation. PPARγ protein is also expressed in Sertoli cells and germ cells (spermatocytes). Its activation can be induced during capacitation and the acrosome reaction. This mini-review will focus on how PPARγ signaling may affect fertility and sperm quality and the potential reversibility of these adverse effects. PMID:25851655

Anabolic steroid use among students at a British college of technology.

PubMed Central

Williamson, D J

1993-01-01

To determine the rate of current or previous use of anabolic steroids by students at a UK college of technology, a questionnaire survey of 687 day students was conducted. The questionnaire began with a general section for all of the students, which ended with the question 'Have you ever used anabolic steroids?'. A further section specifically for anabolic steroid users examined patterns of use, and how certain circumstances might affect the individual's decision to use anabolic steroids. The response rate to the questionnaire was 92%. The overall rate of current or previous use of anabolic steroids was 2.8% (4.4% in males, 1.0% in females). Of these, 56% had first used anabolic steroids at age 15 or less. Anabolic steroid users were more likely to be male, under 17 years of age, and participating in bodybuilding, weight-lifting or rugby. The results of this survey, if confirmed in other groups of young people, would suggest that use of anabolic steroids may be widespread in the UK. PMID:8242280

Anabolic steroid use among students at a British college of technology.

PubMed

Williamson, D J

1993-09-01

To determine the rate of current or previous use of anabolic steroids by students at a UK college of technology, a questionnaire survey of 687 day students was conducted. The questionnaire began with a general section for all of the students, which ended with the question 'Have you ever used anabolic steroids?'. A further section specifically for anabolic steroid users examined patterns of use, and how certain circumstances might affect the individual's decision to use anabolic steroids. The response rate to the questionnaire was 92%. The overall rate of current or previous use of anabolic steroids was 2.8% (4.4% in males, 1.0% in females). Of these, 56% had first used anabolic steroids at age 15 or less. Anabolic steroid users were more likely to be male, under 17 years of age, and participating in bodybuilding, weight-lifting or rugby. The results of this survey, if confirmed in other groups of young people, would suggest that use of anabolic steroids may be widespread in the UK.

Effect of composting on the fate of steroids in beef cattle manure.

PubMed

Bartelt-Hunt, Shannon L; Devivo, Shannon; Johnson, Leslie; Snow, Daniel D; Kranz, William L; Mader, Terry L; Shapiro, Charles A; van Donk, Simon J; Shelton, David P; Tarkalson, David D; Zhang, Tian C

2013-07-01

In this study, the fate of steroid hormones in beef cattle manure composting is evaluated. The fate of 16 steroids and metabolites was evaluated in composted manure from beef cattle administered growth promotants and from beef cattle with no steroid hormone implants. The fate of estrogens (primary detected as estrone), androgens, progesterone, and the fusarium metabolite and implant α-zearalanol was monitored in manure compost piles. First-order decay rates were calculated for steroid half-lives in compost and ranged from 8 d for androsterone to 69 d for 4-androstenedione. Other steroid concentration data could not be fit to first-order decay models, which may indicate that microbial processes may result in steroid production or synthesis in composting systems. We demonstrate that composting is an effective strategy to remove steroid hormones from manure. Total steroid hormone removal in composted beef cattle manure ranged from 79 to 87%. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Oral steroids alone or followed by intranasal steroids versus watchful waiting in the management of otitis media with effusion.

PubMed

Hussein, A; Fathy, H; Amin, S M; Elsisy, N

2017-10-01

To evaluate the effects of oral steroids alone or followed by intranasal steroids versus watchful waiting on the resolution of otitis media with effusion in children aged 2-11 years. A total of 290 children with bilateral otitis media with effusion were assigned to 3 groups: group A was treated with oral steroids followed by intranasal steroids, group B was treated with oral steroids alone and group C was managed with watchful waiting. Patients were evaluated with audiometry and tympanometry. The complete resolution rates of otitis media with effusion were higher in groups A and B than in group C at six weeks. There were no significant differences in otitis media with effusion resolution rates between the groups at three, six and nine months. Oral steroids lead only to a quick resolution of otitis media with effusion, with no long-term benefits. There was no benefit of using intranasal steroids in the management of otitis media with effusion.

Adrenal activity in maned wolves is higher on farmlands and park boundaries than within protected areas.

PubMed

Spercoski, Katherinne M; Morais, Rosana N; Morato, Ronaldo G; de Paula, Rogério C; Azevedo, Fernanda C; May-Júnior, Joares A; Santos, Jean P; Reghelin, Angela L; Wildt, David E; Songsasen, Nucharin

2012-11-01

In this study we measured excreted fecal corticoid metabolites (FCM) in maned wolves (Chrysocyon brachyurus) living within a protected reserve, on farmlands or in a boundary zone between the two habitats, and determined the impacts of season and reproductive status on adrenal activity. Feces were collected within a national park (n=191 samples), a park boundary zone (n=39) and on nearby farmlands (n=27), processed and analyzed by enzyme immunoassay. FCM amounts from samples collected on farmlands were higher (P<0.05) than in those collected inside the reserve and from the boundary zone. In relation to seasonality, FCM were elevated (P<0.05) in spring (September-November) when wolf pairs were raising young. We then divided the samples collected during breeding season (March-August) into cycling females and male/non-cycling females based on fecal progesterone: fecal testosterone ratio. FCM concentrations of the former collected inside the park were higher than (P<0.05) than the latter group. However, there were no differences in FCM levels between the two groups for samples collected in the boundary zone and on farmlands. Furthermore, FCM concentrations of male/non-cycling females samples collected on farmlands were 2- to 5-fold higher (P<0.05) than in counterparts collected inside the park. The consistently high FCM concentrations in samples collected on farmlands indicate that, in addition to seasonality, gender and reproductive status, anthropogenic pressures also contribute to elevating adrenal steroid for individuals living in altered habitat. Copyright © 2012 Elsevier Inc. All rights reserved.

Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins.

PubMed

Christians, Stefan; van Treel, Nadine Denise; Bieniara, Gabriele; Eulig-Wien, Annika; Hanschmann, Kay-Martin; Giess, Siegfried

2016-07-01

Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Nutritive value and qualitative assessment of secondary compounds in seeds of eight tropical browse, shrub and pulse legumes.

PubMed

Babayemi, O J; Demeyer, D; Fievez, V

2004-01-01

Seeds of four tropical multipurpose trees (Albizia saman, Albizia lebbeck, Albizia rhizonse, Leucaena leucocephala), two shrubs (Tephrosia candida, Tephrosia bracteolata) and two pulse legume (Lablab purpureus, Canavalia ensiformis) were chemically analysed for dry matter (DM), ash, crude protein (CP), neutral detergent fibre (NDF) and ether extract (EE). Qualitative evaluation of secondary metabolites (saponins, phenols, steroids, and alkaloids) was elucidated. The DM, ash, CP, NDF and EE ranged between 88.9-93.6 %, 3.0-5.4 %, 24.8-38.2 %, 22.1-46.9 % and 2.0-17.0 % respectively. All seed species contained at least one group of secondary plant metabolites and steroids were common to all except C. ensiformis that was not implicated for any. A. lebbeck and A. rhizonse showed low saponin content. Indications for water soluble tannins were reported for L. leucocephala while the two species of Tephrosia contained flavonoids or condensed tannins. The study suggested the potentials of the legumes seed species as a feed source for ruminants.

Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development

PubMed Central

de Calais, Flávia Leme; Soardi, Fernanda Caroline; Petroli, Reginaldo José; Lusa, Ana Letícia Gori; de Paiva e Silva, Roberto Benedito; Maciel-Guerra, Andréa Trevas; Guerra-Júnior, Gil; de Mello, Maricilda Palandi

2011-01-01

The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT), and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2) was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD) for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency. PMID:22272144

Identification and Characterization of the Genes and Enzymes Belonging to the Bile Acid Catabolic Pathway in Pseudomonas.

PubMed

Luengo, José M; Olivera, Elías R

2017-01-01

The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and other steroids (e.g., testosterone and 4-androsten-3,17-dione). From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemical defined media containing some cyclopentane-perhydro-phenantrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the 9,10-seco pathway, the route involved in the aerobic assimilation of steroids. In this manuscript, we describe the most relevant methods required for (1) isolation and characterization of these species; (2) determining the chromosomal location, nucleotide sequence, and functional analysis of the catabolic genes (or gene clusters) encoding the enzymes from this pathway; and (3) the tools employed to establish the role of some of the proteins that participate in this route.