Kristen Laramie
Alternating DNA methylation and histone modifications to regulate gene transcription of luteinizing hormone receptor
Deregulation of G-protein coupled receptors (GPCR) has been associated with several disease states. It has been shown that the derepression of the luteinizing hormone receptor, a GPCR, in adrenal cells can lead to uncontrolled steroidogenesis and subsequently to toxic levels of hormones such as cortisol. The focus of this research is on the human disease known as Cushing’s disease, in which abnormalities in adrenal GPCRs, including LHR overexpression, is correlated with excess cortisol release. Previous research on human choriocarcinoma cells, human mammary gland carcinoma cells, and human placental cells has shown that coordinated changes in the DNA of the LHR promoter region coupled with histone modifications is involved in regulating silencing of LHR gene transcription. In the present study we have investigated a similar mechanism with human adrenal carcinoma cells, in which we attempted to inhibit silencing of LHR transcription with drugs that create similar coordinated changes in the DNA sequence specifically at the LHR locus. Such in vitro modifications may be useful in identifying possible mechanisms by which the LHR gene becomes derepressed in pathogenesis of Cushing’s syndrome.
Tabitha Manero
Investigation of Signal Transduction for an FSH Receptor Mutant
Follicle-stimulating hormone (FSH) is a peptide hormone that is synthesized in and secreted by the gonadotropes of the anterior pituitary gland. FSH acts in reproduction by stimulating the maturation of germ cells. In the Sertoli cells of the testes, FSH stimulates spermatogenesis. In the ovaries, FSH stimulates the maturation of follicles as well as the production of estrogen. Manipulating FSH activity could lead to treatments for infertility as well as contraceptive uses. In previous experiments it was shown that the mutation of the FSH receptor (K555R) altered the cell-surface residency time. What was unknown was whether this altered cell-surface residency has a correlation with altered signal transduction capability. Our hypothesis was that there would be a decrease in signal transduction. This mutation of the mutation FSH receptor is located in the putative G-protein binding domain, making it likely that it would disrupt the signaling. HEK 293 cells were transfected with wildtype and mutant receptors and then treated with FSH and cAMP production was measured. This is the first experiment to investigate the link between cell surface residency and signal transduction.
Jason Ortolano
Ubiquitination of the Follicle Stimulating Hormone Receptor
An intricate component of female reproductive endocrinology, Follicle Stimulating Hormone (FSH) is responsible for maturation of ovarian follicles and, in conjunction with Leutenizing Hormone (LH), induces ovulation in women by means of a G-protein-coupled receptor. In vitro fertilization (IVF) requires treatment with exogenous FSH to stimulate multiple follicular developments before fertilization can occur. Over stimulation, however, of the ovarian follicles is a potential health risk associated with exogenous stimulation of the follicles via FSH. Women with Polycystic Ovary Syndrome (PCOS) are predisposed to developing Ovarian Hyperstimulation Syndrome (OHSS), which is the health risk associated with exogenous FSH stimulation. As a result of in vitro fertilization via exogenous FSH stimulation, multiple follicle stimulation occurs and can lead to OHSS. Clinicians need a means for minimizing the risk of OHSS and ubiquitination of the receptor has that potential. Cohen, et al. demonstrated that ubiquitination of FSH-containing cells occurs, but was unable to distinguish between cell-surface ubiquitination as a means of receptor desensitization or intracytoplasmic ubiquitination, which would operate as a means of protein quality control to ensure properly folded surface receptors. To distinguish between the different sites of ubiquitination, we will first label the membrane FSH receptors with an anti-body tag and then lyse the cells following ubiquitination. Assaying for the anti-body tag, we will unequivocally distinguish between cellular membrane ubiquitination and intracytoplasmic ubiquitination. We hypothesized that ubiquitination of follicle stimulating hormone target cells occurs at the cell surface. Understanding proper hormonal signal regulation can help fine tune ovarian stimulation for IVF to reduce the risk of developing OHSS.
Alexandra Paul
Determination of species identity of a previously uncharacterized Bacillus subtilis bacterium and the characterization of its antifungal properties
A previously uncharacterized bacterium with potent antifungal properties was determined to be a strain of Bacillus subtilis through sequencing of a portion of its’ 16sRNA. The corresponding gene of a standard ATCC Bacillus subtilis strain was also sequenced and was compared to that of our antifungal strain. The antifungal compound was found to be secreted and capable of passing through a 0.22mm membrane. In addition, I have determined that the activity is stable at 75°C. Production of the antifungal compound appears to be very dependent on growth conditions, potentially due to the effects of quorum sensing. The current assay for detection of the antifungal compound involves inoculation and growth of the bacteria on a sterile O.22 mm filter and subsequent removal of the filter and inoculation of the fungus. The antifungal compound has been shown to be effective against the two fungi tested, Aspergillus niger and Schizphyllum commune. Organic solvent extractions of the agar are presently being performed and will allow for further classification of the antifungal substance. Ongoing studies include the use of gas chromatography-mass spectrometry and high performance liquid chromatography.
Sarah Shea
Investigation of the G-protein coupled receptor (GPCR) spare receptor hypothesis
Human Follicle-stimulating hormone (FSH) acts mechanistically by a GPCR, known as the follicle stimulating hormone receptor (FSHR). In females, FSH plays a crucial role in the early development of ovarian follicles, and in males FSH is necessary for spermatogenesis. FSH is required to stimulate the hormonal changes that lead to ovulation. The signaling action of FSH reaches peak activity and plateaus long before FSH binding activity reaches its peak. This phenomenon has lead to the development of the Spare Receptor Hypothesis; which states that signaling of the follicle stimulating hormone receptor FSHR peaks before binding capacity peaks by desensitization of unoccupied receptors. Using the Spare Receptor Hypothesis as the basis for research we are investigating the mechanism by which signaling potential is maximized before the binding capacity of the receptors on the cell. Polymerase Chain Reaction (PCR) was used to clone a mutant FSHR deficient of the extracellular domain (ECD). This was was transfected into HEK-293 and HEK-293R cells. Western blotting and immunofluorescense verified the presences of the mutant receptor in the HEK cells. Immunopreciptation and phosphorylation studies of the transfected cells will be performed to characterize the activation state of the mutated receptors.
Jonathan Young
Transducing Signals Through the Follicle-Stimulating Hormone Receptor
The follicle stimulating hormone (FSH) is an important factor that promotes oocyte maturation in females and spermatogenesis in males. The desensitization of the FSH receptor is important in regulating signaling pathways associated with FSH. When the receptor binds FSH, it sends a signal that increases cAMP levels in the cell. However, cAMP levels plateau before all the receptors are occupied, suggesting that the cell desensitizes the receptors to the binding of FSH. Phosphorylation provides a potential mechanism through which unoccupied receptors are desensitized. After FSH binds to its receptor, kinases may phosphorylate both the receptor that bound FSH and nearby receptors, turning them off. It is believed that the localization of the receptor in lipid rafts allows for this process to occur. To investigate whether the FSH receptor localizes in lipid rafts, the receptor was immunoprecipitated to see if it binds to caveolin, a marker for caveolae (a special type of lipid raft). An examination of the amino acid sequence of the FSH receptor reveals that it contains a caveolin-binding domain, suggesting the receptor may localize in caveolae. Additionally, immunofluorescence microscopy will be utilized to see if the caveolin co-localizes with the FSH receptor.
Honorary Members:
- Mark Aquino
- Katie Lee- Advisor: Prof. Tom Andersen, Albany Medical College
You must be logged in to post a comment.