Transcript Slide 1
Title Goes Here: Can Be Long or Short Subtitles are fine too. If your title is short you can stick images on either side. DISCUSSION RESULTS INTRODUCTION Table 1: Soluble Protein in Individual Connectives and Ganglia of Intact and Severed VNC’s In this study we investigated ubiquitin, HSC70, and HSP70 in intact and severed ventral nerve cords of the crayfish, Procambarus Clarkii. Ubiquitin is a highly conserved protein that is found in all eukaryotic cells. The ubiquitin-proteasome pathway plays a very important part in the active break down of specific proteins (Ehlers, 2004). Ubiquitin, a 76 amino acid polypeptide, serves as a degradation signal within the pathway in which proteasomes actively degrade certain proteins (Pickart, 2004). The pathway begins with ubiquitin attaching multiple copies of itself to a protein, marking it for degradation. A series of enzymes are involved in the multi-ubiquitination of this pathway. The enzyme E1 is what first activates the ubiquitin molecules. E1 is then followed by E2, which serves to conjugate ubiquitin. The E3 enzyme is a ligase for ubiquitin. Once this is complete, the protein has now been marked for degradation, allowing the 26S proteasome to begin its degradation function (Hicke and Dunn 2003). The proteasome uses ATP and proteases to hydrolyze the protein that has been marked for degradation. The ubiquitin molecules are not broken down in this process because deubiquinating enzymes cause them to release from the marked proteins in the nick of time (Ehlers, 2004; Pickart, 2004). Connective Ganglion Connective Ganglion Connective A1-A2 (µg) A2 (µg) A2-A3 (µg) A3 (µg) A3-A4 (µg) Control 1.5 1.7 1.8 0.3 ----6-21 Control 1.3 0.2 2.3 0.2 0.9 6-22 Control 2.2 ----0.45 0.7 1.85 5-26 Severed 1 day 6-23 Gel #2 Severed 5 days 6-27 3.6 1.7 2.1 0.7 1.0 1.6 3.2 1.9 1.3 1.7 Table 1: The total amount of protein (µg) loaded in each of the samples. Heat shock-stress proteins (HSP’s) and heat shock cognate proteins (HSC’s) are two types of molecular chaperones that can aid the ubiquitin-proteasome pathway with various protein situations (Goldbaum and Landsberg 2004). Both these chaperones assist with protein folding and refolding, as well as targeting irregular proteins for the ubiquitin-proteasome pathway. Within this experiment, we were able to analyze individual connectives and ganglia in severed and non-severed VNC samples. The samples were examined for three different proteins, ubiquitin, HSC70, and HSP70. In Figures 1-5, severed and non-severed samples both contained the proteins HSC70 and HSP70. While in Figure 6, though the ubiquitin protein was present, it was somewhat difficult to identify because of its low intensity on the blot. The identification of the proteins found in Figures 1-5 of our severed and non-severed samples, may assist in further studies on the mechanisms for VNC and MGA survival post severance. It is already known that HSC70 and HSP70 are able to fold and unfold proteins in non-severed axons, and as you can clearly see, even after 9 days post severance (Fig. 5), these proteins are still present in the individual connectives and ganglia. The intensity of these immunoreactive bands may be slightly more intense at the severance site. HSC70 and HSP70 may stabilize unfolded proteins and help them to refold after axonal severance. REFERENCES Batulan, Z., Shinder, G.A., Minotti, S., He, B.P., Doroudchi, M.M., Nalbantoglu, J., Strong, M.J., Durham, H.D. “High Threshold for Induction of the Stress Response in Motor Neurons Is Associated with Failure to Activate HSF1.” The Journal of Neuroscience. 23 (2003): 5789-5798. METHODS & PROCEDURES Ehlers, Michael D. “Deconstructing the axon: Wallerian degeneration and the ubiquitin-proteasome system.” Trends in Neurosciences. 27.1 (2004): 3-6. Acclimation of crayfish to 22°-23°C. Axonal severance was done between the A1 and A2 ganglia. Control VNC’s were left intact. Goldbaum, O. and Richter-Landsberg, C. “Proteolytic Stress Causes Heat Shock Protein Induction, Tau Ubiquitination, and the Recruitment of Ubiquitin to Tau-Positive Aggregates in Oligodendrocytes in Culture.” The Journal of Neuroscience. 24 (2004): 5748-5757. Dissection of VNC in cold Van Haraveld’s Solution (4°C). Individual connectives and ganglia were separated and homogenized individually in lysis buffer. Homogenates were microfuged and diluted with sample buffer. A Bradford Assay was then performed to determine the amount of protein (Table 1). SDS-PAGE was performed, comparing the intact samples versus the severed samples. In order to move proteins from the gel to nitrocellulose, a Western Transfer was performed. Nitrocellulose was then allowed to incubate in Blotto. Nitrocellulose was washed with TBS-T to remove Blotto and then incubated with the primary antibody (mouse anti-ubiquitin or mouse anti-HSP70/HSC70). After incubation, another wash with TBS-T was done and the nitrocellulose was incubated with the secondary antibody (goat antimouse conjugated to alkaline phosphatase). Following another TBS-T wash, nitrocellulose under went an Immunostar treatment. Nitrocellulose was exposed to x-ray film and developed. Proteins collected in the samples of the control VNC’s and the severed VNC’s both recognized the antibodies that were used. In Figures 1-5, the VNC proteins incubated with HSC70/HSP70 antibody contained immunoreactive bands at the predicted molecular weights of mammalian HSC70 (73 kDa) and HSP70 (72 kDa). HSC70 and HSP70 appeared to be common proteins throughout the entire crayfish VNC. With the ubiquitin antibody (Fig. 6), VNC proteins contained an immunoreactive band at the predicted molecular weight of ubiquitin in other systems (8 kDa), but the intensity of the bands was faint. We were able to confirm that crayfish, VNC tissue, as well as VNC proteins are able to survive the surgery of complete axonal severance for at least 9 days (Fig. 5). In order to seal the severed abdominal integument of the crayfish, liquid band-aids were sufficient as proven by the survival of the crayfish. Two other experimental designs were performed along with the primary severance experiment. Instead of severing the VNC at only A1-A2, we also severed the VNC at A3-A4 (data pending). ACKNOWLEDGEMENTS Thanks to Southwestern University’s Fleming Fund for funding the 2005 Biology Summer Research Program Dr. Sheller for her leadership, guidance, and patience throughout this experiment My wonderful lab partner, Angela Nordin, for her constant help in and out of lab. Thanks for holding me down! Everyone involved in the BSRP.