Proteomics & Genomics/General Biochemistry

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    (2014-03) Chib, Rahul; Raut, Sangram; Sabnis, Sarika; Singhal, Preeti; Gryczynski, Zygmunt; Gryczynski, Ignacy
    Ethidium Bromide (EB) is a commonly used dye in a deoxyribonucleic acid (DNA) study. Upon an intercalation, this dye significantly increases its brightness and lifetime. In this report we studied time-resolved fluorescence properties of EB fluorophore existing simultaneously in free and bound forms in the solution. Fluorescence intensity decays were fitted globally to a double exponential model with lifetimes corresponding to free (1.6ns) and bound (22ns) forms, and molar fractions were determined for all used solutions. Anisotropy decays displayed characteristic time dependence with an initial rapid decline followed by recovery and slow decay. This is because of the two existing fractions contributing to a total anisotropy change in time. The short-lived fraction associated with the free EB molecules decreases faster the than the long-lived fraction associated with EB bound to DNA. Consequently, contribution from fast rotation leads to initial rapid decay in anisotropy. On the other hand bound fraction, due to slow rotation helps recover anisotropy in time. This effect of associated anisotropy decays in systems such as EB free/EB-DNA is clearly visible in a wide range of concentrations, and should be taken into account in polarization assays and biomolecular dynamics studies. Purpose (a): To study the interaction of DNA with ethidium bromide. Methods (b): 1) Steady state fluorescence intensity measurement. 2) Time resolved measurements. 3) Steady state anisotropy. Results (c): Increase in fluorescence lifetime and fluorescence anisotropy when ethidium bromide interact with DNA. Conclusions (d): We believe that assays involving EB and DNA should be analyzed with the associated decay model. Neglecting this type of decay pattern can lead to false interpretation of results.
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    (2014-03) Sahyouni, Fatima; Szarka, Szabolcs; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo
    Endocrine disrupting chemicals (EDCs) are a class of chemicals that interfere with the biological actions of hormones, and there has been significant public concern about EDCs in the environment adversely affecting both wildlife and human health. They can alter processes regardless of whether they are related to reproduction. The mammalian uterus is one of the most sensitive organs for estrogenicity, but the widely used uterotrophic rat assay to assess known and potential EDCs merely considers the uterine weight gain endpoint. In this presentation, we focus on the quantitative proteomic investigation of estrogenic endocrine disruption in the rat uterus utilizing a comprehensive systems biology approach. Using 17β-estradiol (E2), an endogenous estrogen, will serve as a reference for subsequent studies of agents with estrogenic potential. Purpose (a): To validate potential markers of estrogenicity of discovery identified differentially expressed estrogen-induced proteins in rat uterine tissue using quantitative proteomics. Methods (b): Ovariectomized rats were treated short-term with subcutaneous E2 injections using corn oil as a vehicle. Approximately 10 mg of tissue were dissected from the uterus of vehicle-treated control and E2-treated animals for proteomic analyses. Uterine proteins were extracted with 8M urea for 30 minutes and subsequently processed by reduction, alkylation and digestion for mass spectrometry analysis. The samples were analyzed using a hybrid linear ion trap–Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source and connected to a nanoflow liquid chromatography system. MS/MS data was searched against a composite UniProt rat protein database using the Mascot software. Quantitation was performed using an MS-based total precursor intensity approach using the Scaffold software. Additionally, the differentially expressed proteins were mapped to signaling networks and biological processes using Ingenuity Pathway Analysis (IPA). Results (c): The mammalian uterus increases its weight due to fluid imbibition and cell proliferation by exogenously administered estrogenic compounds. With the observation of weight gain in the treated uterus compared to non-treated control rats, we confirmed E2’s uterotrophic effects for our subsequent proteomics study. Estrogen-regulated proteins were identified using an MS-based label-free quantitative approach. With p<0.05 considered statistically significant and >2-fold change as threshold, 135 proteins were differentially regulated by the hormone. Of these significantly differentially regulated proteins, 97 were up-regulated in E2-treated uteri and 38 were down-regulated in E2-treated uteri. When these 135 proteins were submitted for bioinformatic pathway analysis, 131 proteins were mapped into 14 networks that merged into E2-regulated pathways. Major molecular processes involve metabolic pathways, steroid signaling, and inflammatory signaling. Top networks include post-translational modification, protein folding, carbohydrate metabolism, cell death and survival, cancer, and endocrine system disorders. Implicated diseases include endocrine system and metabolic disorders. Proteotypic peptides from proteins that were strongly influenced by E2 administration have been selected for targeted validation studies. Conclusions (d): In addition to confirming the expected increase in wet uterine weights, we have derived interaction networks that mechanistically dissect E2’s uterotrophic effect at the proteome level. We have selected proteotypic peptides of strongly regulated proteins for future targeted validation as a potential biomarker panel for estrogenicity. (Supported by the Robert A. Welch Foundation, BK-0031, and the NIH grant AG031535).