Adam graduated in May and is now exploring the mountains of Seattle. Contact information coming soon.
IMD 3 Summer Fellowship Report 2004
Project Title: Detection of Estrogen Mimics in Caribbean Rainforest Botanicals Using Recombinant Estrogen Receptors and Electrophoretic Mobility Shift Assays
Student : Adam J Russell, Trinity University , San Antonio , Texas
Mentor : Dr. James L. Wittliff, Hormone Receptor Laboratory, Department of Biochemistry & Molecular Biology
Throughout the course of this Summer Fellowship I worked under the supervision of Alan Kerr, graduate student in Dr. Wittliff's laboratory and along side Elizabeth Nicholson, fellow IMD 3 scholar. In order to evaluate estrogen-like activities in samples, human recombinant estrogen receptor (rhER) recognition of ERE sequences were evaluated with electrophoretic mobility shift assays (EMSA) that were used to evaluate aqueous and ethanol extracts of medicinal botanicals used by the Carib Indians on the island of Dominica. Botanicals used were Cecropia peltata ( Moraceae ), Eleutherine bulbosa ( Iriaceae ), Momordica charantia ( Cucurbitaceace ), Picrasma excelsa ( Simaroubaceae ), Stachytarpheto jamaicensis ( Verbenaceae ) and Wedelia trilobata ( Asteraceae). Prior to my project, the stems and leaves from these plants were extracted in either absolute ethanol or a phosphate buffer, 50 mM, pH 74 containing 1.5 mM EDTA, 10 mM sodium molybdate and 10% glycerol. Several extracts exhibited potent activities for disrupting rhER recognition of [32P]VitA2 ERE (estrogen response element sequence) when tested using EMSA. These preliminary results suggest that plants used by Carib Indians for so-called “women's problems” contain compounds which influence the mechanism of estrogen activity.
The estrogen receptors (ER a and ER b) play a crucial role in cell proliferation, differentiation, and development. They do this by altering the transcriptional activity of specific genes. Once an estrogenic ligand binds to the ER, it dimerizes and migrates to the nucleus, where it binds to specific DNA sequences (estrogen response elements or EREs). These EREs lie upstream from promoter regions where RNA polymerase (Pol II) can bind and begin transcription. Once the dimer has bound to the ERE, it recruits a co-activator protein which creates a link to the polymerase, allowing it to bind to the promoter sequence more tightly and therefore enhancing transcription.
A method of testing the receptors' affinity for a certain ERE is the EMSA. The EMSA uses a native, non-denaturing, gel, which was loaded with a short radio-labeled DNA sequence (the ERE) that was combined with an already dimerized form of the ER-ligand complex, as well as a particular botanical extract. This mixture migrated through the gel, which separates molecules according to size (smaller molecules migrate farther down the gel). If the ER has successfully bound to the radio-labeled ERE, then the formation of the ER-ERE complex mentioned above is too large to reach the bottom of the gel. Otherwise the botanical extracts inhibit binding of the ER-ERE sequence resulting in a higher amount of ERE present at the bottom of the gel and an absence of a band. Gels were exposed on a phosphorous screen and read with a Cyclone phosphor imager.
The recombinant ER a that was used in these experiments was prepared in a yeast expression system by Dr. Andrei Smolenkov, Investigator in the Hormone Receptor Laboratory. Using the EMSA analyses, it was determined that various botanical extracts inhibited the DNA-binding capacity of both estrogen receptor a and b isoforms. Collectively the data suggest the presence of compounds that appear to block the recognition of ERE sequences by the zinc finger binding motif of human estrogen receptors.
I am very thankful for being supported in part by a grant from Phi Beta Psi Research Foundation and a Summer Fellowship from the Institute for Molecular Diversity & Drug Design.
