Methods for the Quantification of Histochemical Steroid Binding Assays*

2019 ◽  
pp. 143-160
Author(s):  
Louis P. Pertschuk
Keyword(s):  
Binding Assays ◽  
Steroid Binding

Steroid specificity of the putative DHB receptor: evidence that the receptor is not 11βHSD

1998 ◽  
Vol 275 (1) ◽  
pp. E124-E131 ◽  
Author(s):  
Karen E. Sheppard ◽  
Karen Khoo ◽  
Zygmunt S. Krozowski ◽  
Kevin X. Z. Li
Keyword(s):  
Steroid Receptors ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Rat Colon ◽  
Colonic Crypt ◽  
Binding Assays ◽  
Ovary Cells ◽  
High Affinity ◽  
Steroid Binding ◽  
Crypt Cells

Recently, we identified a novel putative nuclear receptor in colonic crypt cells distinct from both mineralocorticoid receptor and glucocorticoid receptor, with high affinity for 11-dehydrocorticosterone (11-DHB) (33). In the present study, competitive nuclear binding assays demonstrated that this site has a unique steroid binding specificity that distinguishes it from other steroid receptors. Western blot analysis showed the presence of 11β-hydroxysteroid dehydrogenase-2 (11βHSD2) but not 11βHSD1 in colonic crypt cells and showed that 11βHSD2 was present in the nuclear pellet. Differences in steroid specificity between the putative DHB receptor and inhibition of 11βHSD activity indicate that binding is not to the enzyme. Furthermore, modified Chinese hamster ovary cells transfected with the 11βHSD2 gene express nuclear 11βHSD2 but not a nuclear DHB binding site. In conclusion, these data support the existence of a novel nuclear DHB receptor in rat colon that is distinct from the classic steroid receptors and from both 11βHSD1 and 11βHSD2.

scholarly journals 11β-Hydroxysteroid Dehydrogenase 1 Transforms 11-Dehydrocorticosterone into Transcriptionally Active Glucocorticoid in Neonatal Rat Heart

Endocrinology ◽  
2002 ◽  
Vol 143 (1) ◽  
pp. 198-204 ◽  
Author(s):  
Karen E. Sheppard ◽  
Dominic J. Autelitano
Keyword(s):  
Cardiac Myocytes ◽  
Rat Heart ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Binding Assays ◽  
Kinase Gene ◽  
Cellular Expression ◽  
Neonatal Rat Heart ◽  
Steroid Binding ◽  
Ribonuclease Protection

Abstract The ability of cells to directly respond to glucocorticoids and aldosterone is a function of GR and MR expression, and coexpression of 11β-hydroxysteroid dehydrogenases (11βHSDs), which convert glucocorticoids and their 11-ketometabolites into either receptor inactive or active derivatives. The aim of the present study was to determine the cellular expression of GR, MR, 11βHSD1, and 11βHSD2 in neonatal rat heart and determine the role these enzymes play in modulating glucocorticoid and aldosterone action. Ribonuclease protection analysis and steroid binding assays showed that GR is expressed in both cardiac myocytes and fibroblasts, whereas MR is expressed only in myocytes. 11βHSD2 was not detected in cardiac cells, but 11βHSD1 was expressed at high levels in both cardiac myocytes and fibroblasts. Enzyme activity studies demonstrated that 11βHSD1 acted as a reductase only, converting biologically inactive 11-dehydrocorticosterone to corticosterone, which then stimulated serum and glucocorticoid-induced kinase gene transcription via GR. In both cardiac myocytes and fibroblasts, aldosterone stimulated serum and glucocorticoid-induced kinase gene expression exclusively via GR, but not MR, indicating that aldosterone can have glucocorticoid-like actions in heart. The ability of cardiac cells to use both circulating corticosterone and 11-dehydrocorticosterone as a source of glucocorticoid suggests that the heart is under tonic glucocorticoid control, implying that glucocorticoids play important homeostatic roles in the heart.

Immunocytochemical localization of corticosteroid-binding globulin in rat tissues

1986 ◽  
Vol 108 (1) ◽  
pp. 31-NP ◽  
Author(s):  
R. W. Kuhn ◽  
A. L. Green ◽  
W. J. Raymoure ◽  
P. K. Siiteri
Keyword(s):  
Binding Sites ◽  
Rat Tissues ◽  
Positive Staining ◽  
Immunocytochemical Localization ◽  
Binding Assays ◽  
Rat Serum ◽  
Paraventricular Nuclei ◽  
Corticosteroid Binding Globulin ◽  
Steroid Binding ◽  
Convoluted Tubules

ABSTRACT Previous studies utilizing steroid-binding assays have suggested that corticosteroid-binding globulin (CBG)-like glucocorticoid binding sites are present in various tissues of the rat. It is not known, however, whether such binding reflects the intracellular presence of CBG derived from serum or a special class (type III) of receptors. In order to elucidate this problem, immunocytochemical localization of rat CBG was carried out using a specific antiserum prepared against rat serum CBG and the peroxidase-antiperoxidase technique. Positive staining was found in certain cells of the liver, the distal and/or convoluted tubules of the kidney, the uterus, the follicular cells of the thyroid, and some cells of the anterior pituitary. Other tissues including heart, muscle, thymus, hypothalamus, supraoptic and paraventricular nuclei, and diaphragm were negative. The presence of immunoreactive CBG in specific cells of some glucocorticoid-responsive tissues and not others raises interesting questions concerning the transport of glucocorticoids and their mechanism of action. J. Endocr. (1986) 108, 31–36

A progesterone binding component in porcine ovarian follicular fluid

1982 ◽  
Vol 94 (1) ◽  
pp. 69-76 ◽  
Author(s):  
A. D. Fleming ◽  
R. W. McGaughey
Keyword(s):  
Steroid Hormone ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Binding Assays ◽  
Follicular Maturation ◽  
Preovulatory Follicles ◽  
Steroid Binding ◽  
Ovarian Follicular Fluid ◽  
Follicular Fluids ◽  
Binding Component

Steroid hormone concentrations (progesterone, oestrone, oestradiol-17β) in porcine follicular fluids were measured by radioimmunoassay. In confirmation of several previous studies, as follicular maturation proceeded, the concentrations of all three steroids increased, although the relative amounts of the oestrogens decreased compared to progesterone. Analysis of fluids for steroid-binding components was undertaken using ion-exchange chromatography, gel filtration and steroid-binding assays. Evidence is presented in support of a labile binding component which exhibits high affinity and low capacity for progesterone and little or no affinity for oestradiol. Our findings are discussed in relationship to the known retention of progesterone within preovulatory follicles and to previous reports of steroid-binding components in follicular fluids.

MEASUREMENT OF STEROID BINDING PROTEINS

1970 ◽  
Vol 65 (1_Suppl) ◽  
pp. S104-S121 ◽  
Author(s):  
E. E. Baulieu ◽  
J. P. Raynaud ◽  
E. Milgrom
Keyword(s):  
Kinetic Parameters ◽  
Binding Proteins ◽  
Binding Specificity ◽  
Binding Parameters ◽  
Target Organs ◽  
Biological Mixtures ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
Steroid Binding Protein

ABSTRACT A brief review of the characteristics of steroid binding proteins found in the plasma and in some target organs is presented, followed by some general remarks on binding »specificity« and binding parameters. Useful techniques for measuring binding parameters at equilibrium are reported, both those which keep the equilibrium intact and those which implicate its disruption. A concept is developed according to which the determination of a specific steroid binding protein is based on the »differential dissociation« of the several steroid binding complexes present in most biological mixtures. Methods which allow determination of the kinetic parameters of the binding systems are also presented. Various representations of the binding and therefore different modes of graphic representation and calculation are discussed, including the recent »proportion graph« method.

Foetal steroid binding protein in British and Japanese women

1987 ◽  
Vol 114 (4) ◽  
pp. 584-588 ◽  
Author(s):  
M. Jawed Iqbal ◽  
Alastair Forbes ◽  
Mark L. Wilkinson ◽  
John W. Moore ◽  
Roger Williams ◽  
...  
Keyword(s):  
Ovarian Function ◽  
Binding Protein ◽  
Premenopausal Women ◽  
Japanese Women ◽  
Sex Hormone Binding Globulin ◽  
Partial Purification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Characteristics ◽  
Steroid Binding ◽  
Steroid Binding Protein

Abstract. In order to examine the newly-discovered sex-steroid binding protein, foetal steroid binding protein (FSBP) in different populations, its binding characteristics and its level were studied by two-tier column ligand binding assay and enzyme-linked immunosorbent assay (ELISA) respectively. In 10 Japanese premenopausal women, analysis of 5α-dihydrotestosterone (DHT) binding in the Cibacron Blue 3GA-Sepharose 6B portion of the column showed a rising plateau pattern with a mean maximum binding of 31.1 ± 7.41%, whereas of 9 similar British women, 8 displayed unsaturable, non-cooperative binding of 11.6 ± 8.22% (P < 0.01). After partial purification of FSBP in these samples, the protein exhibited saturable binding kinetics, median binding 25 (interquartiles 23–34) and 19 (13–25) nmol DHT/l in Japanese and British women, respectively (P < 0.05). By analyzing FSBP by ELISA in 56 Japanese (45 premenopausal) and 59 British (25 premenopausal) women, higher levels were obtained in the whole Japanese group (P = 0.0016) and in the premenopausal Japanese women (P = 0.018) than in their British counterparts. In both nationalities, FSBP levels were higher in premenopausal women, and there was a significant negative correlation of FSBP with age in both populations, particularly in postmenopausal women. FSBP levels did not correlate with weight, parity, sex hormone binding globulin or albumin levels. The influence of FSBP on free steroid levels remains unclear, but some relationship with ovarian function seems a possibility.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

Single Amino Acid Based Self-Assemblies of Cysteine and Methionine

2018 ◽  
Author(s):  
Nidhi Gour ◽  
Bharti Koshti ◽  
Chandra Kanth P. ◽  
Dhruvi Shah ◽  
Vivek Shinh Kshatriya ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Self Assembly ◽  
Aromatic Amino Acids ◽  
Neutral Condition ◽  
Single Amino Acid ◽  
Binding Assays ◽  
Human Neuroblastoma ◽  
Cytotoxicity Assays ◽  
First Time

We report for the very first time self-assembly of Cysteine and Methionine to discrenible strucutres under neutral condition. To get insights into the structure formation, thioflavin T and Congo red binding assays were done which revealed that aggregates may not have amyloid like characteristics. The nature of interactions which lead to such self-assemblies was purported by coincubating assemblies in urea and mercaptoethanol. Further interaction of aggregates with short amyloidogenic dipeptide diphenylalanine (FF) was assessed. While cysteine aggregates completely disrupted FF fibres, methionine albeit triggered fibrillation. The cytotoxicity assays of cysteine and methionine structures were performed on Human Neuroblastoma IMR-32 cells which suggested that aggregates are not cytotoxic in nature and thus, may not have amyloid like etiology. The results presented in the manuscript are striking, since to the best of our knowledge,this is the first report which demonstrates that even non-aromatic amino acids (cysteine and methionine) can undergo spontaneous self-assembly to form ordered aggregates.

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