Netrin-1-like Peptides Are Secreted by Tetrahymena thermophila

Netrin-1 is a peptide signaling molecule that has many roles in vertebrates. In the ciliated protozoan, Tetrahymena thermophila, netrin-1 acts as a chemorepellent, causing cells to exhibit a characteristic avoidance behavior. We have previously shown that netrin-1 avoidance by T. thermophila is inhibited by genistein, which is a broad spectrum tyrosine kinase inhibitor. One question we wished to answer in our current study was, “Is genistein specifically acting upon tyrosine kinases in order to inhibit netrin avoidance in Tetrahymena?” In order to answer this question, we used the phytoestrogen, diadzein, as a negative control for genistein inhibition. Diadzein had no effect on avoidance, suggesting that genistein inhibition is specific. In order to gain more information about the nature of the kinases involved in netrin-1 signaling, we tested several other kinase inhibitors, including a src inhibitor, a focal adhesion kinase inhibitor, and a Rho kinase inhibitor. Each of these kinases has been implicated in netrin-1 signaling in some vertebrate cell types. However, none of these inhibitors affected Tetrahymena avoidance to netrin-1. Finally, we wished to answer the question, “Is netrin-1 actually serving an autocrine signaling role in Tetrahymena, or is the peptide merely serving as an agonist for another receptor?” In order to answer this question, we prepared a whole cell extract of Tetrahymena using 0.1% SDS. We also washed Tetrahymena in our behavioral buffer and allowed them to sit in that buffer for 24 hours. The Tetrahymena were centrifuged out of the buffer, and the supernatant, containing the proteins which the Tetrahymena had secreted, was kept for ELISA assay. An ELISA, using a polyclonal anti netrin-1 antibody, was run on the whole cell extract and the secreted proteins against a netrin-1 standard curve. Both the secreted proteins and the whole cell extract tested positive for netrin-1 in the ELISA. Further experimentation will allow us to determine the nature of these netrin-like peptides

The Effects of a Prescribed Burn on Small Mammals in an Ohio Tallgrass Prairie

Prescribed burning is used on prairies for increased prairie health and to manage the communities of organisms that rely on the prairie’s natural resources. Small mammals are also affected by the burning of prairies. Previous research by Chance in 1986 and Cook in 1950 has established that burning decreases the small mammal capture rates in the burned area because of the habitat loss and rapid change of habitat post-burn that stresses the animals and leads to lower reproductive levels (Chance, 1986). Other studies by Francl and Small also showed that small mammal populations did not return to pre-burn numbers even after 16 months of evaluation (Francl, K. E., & Small, C. J., 2013). We hypothesize that small mammal populations will decrease in the prairie after burning because of a lack of habitat and food source and that they will emigrate from the burning site to nearby prairie or similar habitat. We will use baited Sherman live traps placed at 25 meter intervals in order to estimate the various small mammal populations in each of the observed habitats. We will plot the capture locations on a map of the area in order to track how stress affects the shift of population density

Netrin-1 Peptide Is a Chemorepellent in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrin-1 is a highly conserved, pleiotropic signaling molecule that can serve as a neuronal chemorepellent during vertebrate development. In vertebrates, chemorepellent signaling is mediated through the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2. Tetrahymena thermophila has been used as a model system for chemorepellent signaling because its avoidance response is easily characterized under a light microscope. Our experiments showed that netrin-1 peptide is a chemorepellent in T. thermophila at micromolar concentrations. T. thermophila adapts to netrin-1 over a time course of about 10 minutes. Netrin-adapted cells still avoid GTP, PACAP-38, and nociceptin, suggesting that netrin does not use the same signaling machinery as any of these other repellents. Avoidance of netrin-1 peptide was effectively eliminated by the addition of the tyrosine kinase inhibitor, genistein, to the assay buffer; however, immunostaining using an anti-phosphotyrosine antibody showed similar fluorescence levels in control and netrin-1 exposed cells, suggesting that tyrosine phosphorylation i s not required for signaling to occur. In addition, ELISA indicates that a netrin-like peptide is present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Further study will be required in order to fully elucidate the signaling mechanism of netrin-1 peptide in this organism