Browsing by Subject "Department of Biochemistry, Molecular Biology, and Biophysics"

Now showing 1 - 7 of 7
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    Developing an Anti-Tumor Therapy: Expression of mLIGHT Cytokine in Escherichia coli & Salmonella enterica Typhimurium
    (2012-04-18) Ton, Angie
    Current conventional cancer treatments such as chemotherapy and radiation have many limitations and adverse side effects. Salmonella enterica Typhimurium has been shown to colonize solid tumors, making this bacterium a potentially useful vector for delivering cytokines that promote tumor rejection. The objective of this project is to express mouse LIGHT (mLIGHT) cytokine, a tumor necrosis factor that promotes apoptosis, as a secreted protein in first E. coli and then S. enterica. Like S. enterica, E. coli is a Gram-negative facultative anaerobe, but can be used in a Biosafety Level 1 labtoratory. Soluble proteins, insoluble proteins, and secreted proteins were isolated from E. coli cultures harboring expression vectors that contain mLIGHT cDNA and the hemolysin secretion signal sequence under the control of the Trc promoter. Western blotting indicated that mLIGHT is expressed as an insoluble protein, but not as a soluble or secreted protein. These findings suggest that other promoters and secretion signal sequences need to be tested to express mLIGHT as a secreted protein. Once a construct is made that results in expression of mLIGHT as a secreted protein in E. coli, the construct will be introduced into S. enterica. The bacterium will eventually be evaluated for its effectiveness as an anti-tumor therapy in mouse models of solid tumors.
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    Developing an Anti-Tumor Therapy: Expression of mTRAIL Cytokine in Escherichia coli
    (2012-04-18) Arries, Cade
    The goal of this project is to develop a bacteria-based therapy for cancer. Since conventional radiation and chemotherapies attack cells on the outside of the tumors, another strategy is needed to kill cells in the interior of the tumor. Solid cancerous tumors often have nearly anaerobic cores due to poor blood circulation, and some anaerobic bacteria are known to enter tumor cells and reproduce. If these bacteria can be engineered to produce cytokines that will activate the immune system, then all of the tumor cells could be eliminated. Here we show the successful modification of a bacterial plasmid in Escherichia coli to express and secrete a known human apoptotic factor (TRAIL), which aids in controlled cell death of cancerous cells. Once expression is optimized, this plasmid will be introduced into an attenuated strain of Salmonella enteria Typhimurium, which is known to invade tumor cells. This bacterium can then be tested as a therapy in mouse models of cancer.
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    Development of APOBEC3 Cytosine Deaminase Inhibitors
    (2011-09-27) Grover, Torie; Perkins, Angela; Li, Ming; Harris, Reuben; Harki, Daniel
    The APOBEC3 (A3) family of proteins degrade non-native or ‘foreign’ DNA in cells. We have hypothesized that blocking the enzymatic activity of A3 proteins could enhance the efficiency of foreign DNA introduction (transfection) into cells that are otherwise refractory to the process. APOBEC3 proteins degrade ‘foreign’ DNA by converting cytosines into uracils, which then triggers the cell to degrade the DNA due to the presence of a non-native DNA base (uracil). To identify small molecules that could inhibit A3 proteins, High Throughput Screening (HTS) was performed at the University of Minnesota and the Sanford-Burnham Medical Research Institute and over 350,000 compounds were tested for inhibition of A3A and A3G proteins. Follow-up studies by the Harris laboratory (University of Minnesota) have identifed hundreds of potential candidate molecules that can inhibit A3 activity in vitro. Three lead molecules from this study include MN152, MN184 and MN132. The Harki laboratory (University of Minnesota) is collaborating with the Harris laboratory to conduct detailed medicinal chemistry campaigns to optimize lead molecules for strong potency and minimal toxicity. Preliminary results from our synthesis studies of these chemotypes are presented here.
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    Effect of Experimental Drug S107 on Accessory Protein Binding to the RyR
    (2012-04-18) Popa, Mircea Anton
    The physiological basis of muscle-generated motor activity depends on the precise regulation of intracellular Ca2+ ions. Central to this process is the sarcoplasmic reticulum (SR), a special organelle in muscle cells that is responsible for ion storage. The Ca2+ ions are released from the SR through a large conductance channel known as the ryanodine receptor (RyR). The channel’s activity is in turn regulated by intracellular messengers, including FK506 binding proteins (FKBP12.6 & 12.0), and calmodulin (CaM). The experimental heart failure drug S107 has been associated with improved muscle performance, though it is still unclear if it directly affects the binding activity of either the FKBPs or CaM. Here, the effect of S107 on the binding of fluorescent FKBPs and CaM to RyR is characterized. The results show that S107 slightly, but significantly, decreases the binding affinity of CaM; though, only in nanomolar calcium concentrations, and when the RyR is treated with oxidized glutathione. S107 has no significant effect on the binding of FKBP to RyR. Prior to this research, it was thought that S107 might significantly increase the binding affinity of FKBP. The effect of S107 on CaM binding to RyR has not been previously investigated. These results suggest that the primary mechanism through which S107 achieves its physiological effects does not require or affect FKBP binding. Further studies will have to determine whether the effect of S107 on CaM binding to RyR is linked to the inhibition of Ca2+ leak in the channel.
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    The Search for Novel Cancer Therapies : Catalytic Inhibition of Topoisomerase II by Substituted 9-aminoacridine Derivatives
    (2011-04-13) Etchison, Ryan; Jacobson, Blake; Dixon, Joe-Jay; Kratzke, Robert
    The objective of this research is to test the antiproliferative effects of four novel substituted 9-aminoacridine derivatives on lung cancer and to determine their viability as potential therapeutic agents for use in a clinical setting.
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    Structural Investigation of FtsK and Close Homologs
    (2012-04-18) Klimek, Theodore
    FtsK is an integral membrane DNA translocase of the Fts family found within Escherichia coli and with close homologs across many bacterial species. Some of those homologs (Tra family) are also involved in a variety of type IV secretion systems, commonly involved in bacterial conjugation and often in infection. Our work focused on the cloning, expression, purification, and crystallization of FtsK and several homologs, TrwB, TraD, and TraG, for the purpose solving the atomic resolution structure of these proteins. We prepared a variety of constructs in bacterial T7 based expression vectors for the purpose of expressing these proteins in E. coli. For the proteins that showed positive expression, the protein was solubilized with detergent and then purified through nickel-NTA affinity and size-exclusion chromatography (SEC). Results are shown through SEC reports and SDS-PAGE gels that indicate protein purity (by gel) and homogeneity and oligomeric state (by SEC). Many bacterial membrane based proteins, especially those of FtsK and its relatives are accesible targets for antibiotic intervention in humans as they are unique to bacterial systems. For this reason we hope that the procedures followed here may enable further development of our knowledge of these systems, that we might maintain a leg up on rapidly evolving pathogenic species.
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