Determination of estrogen presence in water by SPR using estrogen receptor dimerization

Estrogenic compounds are a class of pharmaceutical products harmful to animals and a cause of environmental damage. The biological activity of these compounds is high since they have been designed to act at low concentrations. Thus, even at the low concentrations found in the environment, they may produce deleterious effects on aquatic organisms as well as on humans, who might be contaminated in a number of ways (via drinking water or contaminated food, for example). We used the property of these compounds to bind a specific protein (estrogen receptor, ER) to develop a quantification method of these chemical entities. Estrogenic compound detection was performed using ER dimerization properties monitored by surface plasmon resonance (SPR). The ligand-activated ER dimer was detected by its interaction with a specific DNA consensus sequence estrogen response element. The concentration and the nature of the estrogenic compounds modified the SPR signal and were characteristic of the ligand-dependent homodimerization of ER. For 17β-estradiol, dimerization of ER was experimentally determined at an ER to 17β-estradiol ratio near 1:1. Estrogenic compounds (17β-estradiol, estriol, estrone, ethynyl estradiol) activated the dimerization process at different concentration levels, while some others (tamoxiphen, resveratrol, genistein, bisphenol A) did not seem to have any effects on it. We demonstrated that this method allows the direct detection of 17β-estradiol at concentrations above 1.4 μg/L (5 nM).     

Kinetic analysis of actinides using differential spectrophotometry

The kinetic method developed allowed us to analyse of two-component actinide mixtures without separation. The concentration changes in the system where complex dissociation reaction takes place were determined by spectrophotometry.     

Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS)

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique for detecting interactions of molecules immobilized on a surface with ligands in solution. Here we show that RIfS also permits the detection of the adhesion of tissue culture cells to a functionalized surface in a flow system. Interactions of T cells with other leukocytes or epithelial cells of blood vessels are crucial steps in the regulating immune response and inflammatory reactions. Jurkat T cell leukemia cells rapidly attached to a transducer functionalized with a monoclonal antibody directed against the T cell receptor (TCR)/CD3 complex, followed by activation-dependent cell spreading. RIfS curves were obtained for the Jurkat derivative JCaM 1.6 (which lacks the key signaling protein Lck), cells preincubated with cytochalasin D (an inhibitor of actin polymerization), and for surfaces functionalized with an antibody directed against the coreceptor CD28. These curves differed with respect to the maximum signal and the initial slope of the increase in optical thickness. The testing of chemical inhibitors, cell surface molecules and gene products relevant to a key event in T cell immunity illustrates the potential of label-free techniques for the analysis of activation-dependent cell-surface contacts. The first two authors contributed equally to this paper     

An advanced biosensor for the prediction of estrogenic effects of endocrine-disrupting chemicals on the estrogen receptor alpha

A label-free and time-resolved biosensor based on reflectometric interference spectroscopy (RIfS) has been developed to evaluate the agonistic or antagonistic effects of potential ligands with unknown behavior. The biosensor utilizes the specific interaction between the estrogen receptor alpha (ER alpha) and short specific peptides. The unique feature of these peptides allows the investigation of the behavior of ligands and the discrimination between the agonistic and antagonistic effects caused by conformational changes of the receptor. Thus, this developed biosensor allows not only the differentiation between ligands and nonligands of a receptor, but also the potential of these ligands to influence conformational changes in the receptor, leading to activation or inhibition of the receptor-dependent pathways. Owing to the robustness of the direct optical detection principle used, the biosensor is applicable to complex biological matrices, even crude cell extracts. Moreover, the reliability of the biosensor, including regeneration steps when performing subsequent measurements, has been verified.     

Kinetic analysis of catalytic slurry oil pyrolysis using thermogravimetric analysis

Journal of Thermal Analysis and Calorimetry - Pyrolysis is an important process for converting unconventional fuels, such as heavy oil, residues and oil sand, to high-value-added light products....     

Kinetic analysis of complex decomposition reactions using evolved gas analysis

For complex decomposition reactions, traditional methods, such as TG and DSC cannot fully resolve all of the steps in the reaction. Evolved gas analysis (EGA) offers another tool to provide more information about the decomposition mechanism. The decomposition of sodium bicarbonate was studied by TG, DSC and EGA using a simultaneous thermal analysis unit coupled to a FTIR. The decomposition of sodium bicarbonate involves two reaction products H₂O and CO₂, which are not evident from either TG or DSC measurements alone. A comparison of the reaction kinetics from TG, DTG and EGA data were compared.     

Kinetic analysis of wheat straw pyrolysis using isoconversional methods

The pyrolysis of wheat straw has been carried out by means of thermogravimetric analysis in inert atmosphere. The samples were heated over a range of temperatures that includes the entire range of pyrolysis with three different heating rates of 5, 10 and 20 K min⁻¹. The activation energy values as a function of the extent of conversion for the pyrolysis process of wheat straw have been calculated by means of the Flynn–Wall–Ozawa isoconversional method, the Vyazovkin–Sbirrazzuoli isoconversional method and an iterative isoconversional method presented in this article. The results have showed that there are small differences between the activation energy values obtained from the three methods, and the pyrolysis process reveals a dependence of the activation energy on conversion and have indicated the validity of the iterative integral isoconversional method. The effective activation energy for the pyrolysis of wheat straw is 130–175 kJ mol⁻¹ in the conversion range of 0.15–0.85. Furthermore, the prediction of the pyrolysis process under isothermal conditions from the dependence of the activation energy on the extent of conversion has been presented.     

Kinetic analysis of second order reactions using UV-VIS spectroscopy

A new method was recently developed for the spectroscopic kinetic analysis of reactions with two linearly independent concentration variables. Firstly, the results of this theory applied to experimental data are represented. For this investigation, reactions of the mechanism type A + Bk (1)--> C + D and A + E uc-->|k(2)|F + G were chosen since both reaction steps can be studied separately. The evaluation of the reaction system consisting of both reaction steps gives satisfactory results when the methods of "formal integration" and "singular value decomposition" (SVD) are used. Absorption coefficients of absorbing species are not needed for the evaluation. Only time dependent spectroscopic measuring values and initial concentrations are used in the presented practical example.     

Study on interactions of endocrine disruptors with estrogen receptor using fluorescence polarization

In this study, we examined the affinities of many (21) compounds such as hormones, pharmaceuticals, industrial chemicals, and phytoestrogens to the estrogen receptor (ER) by ER binding assay using fluorescence polarization (FP). This method is based on the competitive binding assay using fluorescein-labeled estradiol (F-E2), in which the polarization values decreased with the addition of the compounds (competitors). The obtained sigmoidal inhibition curves were transformed into the pseudo-Hill plots, and the concentrations at 50% inhibition (IC50) and Hill coefficients were obtained from the regression equations. We examined the relationship between the chemical structures and estrogenic activities, and finally classified the tested compounds into three categories, agonists, partial agonists and antagonists based on their Hill coefficients.     

Normal-mode analysis of the glycine alpha1 receptor by three separate methods

Predicting collective dynamics and structural changes in biological macromolecules is pivotal toward a better understanding of many biological processes. Limitations due to large system sizes and inaccessible time scales have prompted the development of alternative techniques for the calculation of such motions. In this work, we present the results of a normal-mode analysis technique based on molecular mechanics that enables the calculation of accurate force-field based vibrations of extremely large molecules and compare it with two elastic network approximate models. When applied to the glycine alpha1 receptor, all three normal-mode analysis algorithms demonstrate an "iris-like" gating motion. Such gating motions have implications for understanding the effects of anesthetic and other ligand binding sites and for the means of transducing agonist binding into ion channel opening. Unlike the more approximate methods, molecular mechanics based analyses can also reveal approximate vibrational frequencies. Such analyses may someday allow the use of protein dynamics elucidated via normal-mode calculations as additional endpoints for future drug design.     

Kinetic analysis of microemulsions using microprocessor controlled UV/visible-spectrometry

Microprocessor controlled spectrometers greatly extend the possibilities for kinetic analysis of complex thermal or photochemical reactions. As a result, more sophisticated evaluation procedures, such as derivative spectroscopy or dynamic multi-component analysis become possible.     

Binary quantitative structure-activity relationship (QSAR) analysis of estrogen receptor ligands

The use of high throughput screening (HTS) to identify lead compounds has greatly challenged conventional quantitative structure-activity relationship (QSAR) techniques that typically correlate structural variations in similar compounds with continuous changes in biological activity. A new QSAR-like methodology that can correlate less quantitative assay data (i.e., "active" versus "inactive"), as initially generated by HTS, has been introduced. In the present study, we have, for the first time, applied this approach to a drug discovery problem; that is, the study of the estrogen receptor ligands. The binding affinities of 463 estrogen analogues were transformed into a binary data format, and a predictive binary QSAR model was derived using 410 estrogen analogues as a training set. The model was applied to predict the activity of 53 estrogen analogues not included in the training set. An overall accuracy of 94% was obtained.     

Kinetic analysis of consecutive reactions using TG and DSC techniques

A complex mechanism of thermal degradation processes was postulated for the reaction type , and a theoretical analysis of DTG and DSC curves was followed by corresponding mathematical simulation. In this scheme, compoundS represents a volatile product (e.g. gas and/or vapor), which is a necessary assumption in order to explain the differences in results which can arise after the kinetic analysis of DTG and DSC experimental data.Mathematical simulation was performed with different values of the Arrhenius parameter for both reaction rate constants (k ₁, andk ₂) in the range 83.1–291.0 kJ/mol for the activation energy and with corresponding values for the frequency factor (10⁵–10¹⁹ min^–1). The influence of endothermic heat effects (50–200 kJ/mol) in the reactionsAE andRS was also investigated. The calculated rates of mass and heat change (DTG and DSC), for different heating rates, showed that the maximum values of the two curves are reached at different temperatures. The non-uniformity of the DTG and DSC maxima depends on the difference between the values of the reaction rate constants and their temperature sensitivities (E ₁ andE ₂) and also on the heating rate.The theoretical analysis performed demonstrated the possibility of determining the reaction rate parameters (k ₁ andk ₂) in the case of consecutive first-order reactions, by using simultaneous TG and DSC analysis.The proposed theoretical analysis is supported by experimental DTG/DSC data concerning the pyrolysis of oil shale, which were interpreted in terms of consecutive reactions.

---

Zusammenfassung Unter Annahme eines komplexen Mechanismus für thermische Abbauprozesse wurde für den Reaktionstyp eine theoretische analyse von DTG- und DSC-Kurven, gefolgt von einer entsprechenden mathematischen Simulation durchgeführt. Hier stellt die VerbindungS ein flüchtiges Produkt dar (lias oder Dampf), was eine notwendige Bedingung zur Erklärung von unterschiedlichen Ergebnissen nach der kinetischen Analyse der experimentellen DTG- und DSC-Daten ist. Bei der mathematischen Simulation wurden unterschiedliche Arrheniusparameterwerte benutzt, für beide Reaktionsgeschwindigkeitskonstanten (k₁ und k₂) in einem Intervall von 83,1–291,0 kJ/mol für die Aktivierungsenergie sowie unter Anwendung entsprechender Frequenzfaktoren (10⁵–10¹⁹ min^–1). Für die ReaktionenA R undR S wurde auch der Einfluß von endothermen Wärmeeffekten (50–200 kJ/mol) untersucht. Die berechneten Geschwindigkeiten für die Veränderung von Masse und Wärme (DTG und DSC) bei verschiedenen Aufheizgeschwindigkeiten zeigen, daß beide Kurven ihr Maximum bei unterschiedlichen Temperaturen erreichen. Die Ungleichmäßigkeit der DTG- und DSC-Maxima basiert auf den unterschiedlichen Werten der Reaktionsgeschwindigkeitskonstanten, deren Temperaturempfindlichkeit (E ₁ undE ₂ und hängt auch von der Aufheizgeschwindigkeit ab. Die durchgeführte theoretische Analyse zeigt, daß es mit Hilfe simultaner DTG- und DSC-Untersuchungen möglich ist, im Falle konsekutiver Reaktionen erster Ordnung die Reaktionsgeschwindigkeitsparameter (k ₁ undk ₂) zu bestimmen. Das vorgeschlagene theoretische Analysenverfahren wird am Beispiel der experimentellen DTG/DSC-Daten der Pyrolyse von Ölschiefer und Anwendung konsekutiver Reaktionen illustriert.

---

. S (. ), , . ( _{1 2}) 83,1–291,0 · ^–1 (10⁵–10^{19 –1}). (50–200 ·^–1) AR RS. ( ) , . (E ₁ E ₂), . , ₁, ₂ . / , .

---

The authors are greatful for financial support to the KFA International Bureau D-5170 Jülich on the German side, and to the Research Fund of the Belgrade Region Business Association for the Exploration, Production and Exploitation of Oil Shales, Belgrade, and SOUR Jugopetrol, Belgrade on the Yugoslavian side.     

Destabilizing domains derived from the human estrogen receptor

Methods to rapidly and reversibly perturb the functions of specific proteins are desirable tools for studies of complex biological processes. We have demonstrated an experimental strategy to regulate the intracellular concentration of any protein of interest by using an engineered destabilizing protein domain and a cell-permeable small molecule. Destabilizing domains have general utility to confer instability to a wide range of proteins including integral transmembrane proteins. This study reports a destabilizing domain system based on the ligand binding domain of the estrogen receptor that can be regulated by one of two synthetic ligands, CMP8 or 4-hydroxytamoxifen.