Biochemistry and Molecular Biology.
During development, basic helix loop helix (bHLH) transcription factors are responsible for guiding cells to a tissue-specific fate and initiating differentiation. Nato 3, a bHLH transcription factor, is expressed in the neural tube of the developing Mus Musculus and Gallus Gallus embryo. Published literature suggests Nato 3 is a transcriptional inhibitor of the neurogenic transcription factor Ascii, suggesting that Nato 3 would be a negative regulator of neurogenesis. The objective of our study was to determine which population of neural progenitor cells in the neural tube expresses Nato 3. We hypothesized that Nato 3 expression is restricted to the floor plate region of the developing neural tube at all axial levels in both the Mus Musculus and Gallus Gallus embryos. We established and optimized the in situ hybridization technique to detect endogenous Nato 3 mRNA expression. Embryos were harvested and sectioned at multiple developmental stages. We found Nato 3 is expressed in the floor plate region of the spinal cord, hindbrain and midbrain at the onset of neurogenesis and continues until late gestation. Most of the cells of the floor plate region serve as a signaling center and do not differentiate into neurons. The expression pattern suggests that Nato 3 may have a role in the function of floor plate cells throughout the course of development. A critical question is to determine if Nato3 promotes the floor plate cell lineage and if this occurs at the expense of neurogenesis.
Using Affinity Tagged Fluorescent Proteins in the Biochemistry Teaching Laboratory. Eric J. Arnoys, Eric Nollet, and Kim Spronk, Calvin College, Dept. of Chemistry and Biochemistry
Though protein purification is fundamental to the biochemistry laboratory, many undergraduates have a difficult time visualizing it. For too many students, each step of the process involves application of an abstract concept, and even with a detailed experimental write up at hand, simple decisions such as whether to keep a supernatant or pellet are not straightforward. Often SDS-PAGE provides the only evaluation of experimental success; when students have a poor gel, even this evaluation fails, and the experimenters have no idea what may have gone wrong. To address these common problems, we have constructed a multi-week protein purification and characterization project centered on fluorescent proteins. In the first laboratory period, each pair of students receives a bacterial lysate containing a pair of unknown fluorescent proteins, one of which possesses an affinity tag. Over the next few weeks, the students use a bevy of common techniques to identify their two unknowns. In the process, students learn about protein overexpression, chromatography, protein and DNA electrophoresis, fluorescence spectra, PCR, plasmid purification, and protein denaturation. We have found that the intense greens, cyans, yellows, oranges, and reds of the overexpressed proteins make abstract concepts come alive.
Capture and Analysis of Unmethylated DNA Using CXXC Domain of MBD 1. Mousab M. Eteer, University of Michigan--Flint, Department of Biology
DNA methylation patterns are of great importance in the human genome. They can be indicative of illnesses and diseases, such as cancer and FMR (Fragile X Mental Retardation Syndrome). This project aimed at creating a capture and analysis matrix for unmethylated CpG DNA. To do this, a certain protein that binds specifically to unmethylated CpG DNA was utilized. CXXC-3 is a domain found in MBD-1 (Methyl binding domain) that hinds to unmethylated CpG. Through adding specific tags to this domain we can purify bound DNA on either a solid phase matrix or a magnetic affinity matrix. This technology will enable us to detect diseases based on one's DNA methylation profile for specific genes. In the project CXXC-3 was tested in four different forums, the main form and three specifically tagged forms. The main form of CXXC-3 was tested for binding. Fluorescently labeled DNA was used and the results analyzed using the GE Typhoon Analyzer. The other CXXC-3 protein constructs were labeled with MBP (Maltose binding protein), HIS-TAG (Histadine Tag), and HIS-TAG-CBD (Histadine Tag, Chitin binding protein). Each of these constructs was tested for binding and then the HIS-TAG-CBD construct was used in an extraction.
Association of the Anillin Related Protein Midi with Actin. Jennifer Jakubowski and Dawn Clifford Hart, Grand Valley State University, Department of Cell and Molecular Biology
Schizosaccharyomyces pombe, like human cells, divide symmetrically through constriction of an acto-myosin ring. The conserved protein Mid1 plays a key role in the early assembly of the ring. Cells lacking midi form acto-myosin rings that are non-centered and disorganized, resulting in unequal division of the cellular contents. In human cells, a Midi related protein called anillin regulates contraction of this ring. Anillin contains an actin binding domain that is necessary for the protein's direct association with actin. Unlike anillin, Midi does not contain an actin binding domain, and its ability to interact directly with actin has not been previously reported. However, previous studies show that anillin depleted and Midi depleted cells show similar defects in ring placement, ring formation, and cytokinesis. To assess whether or not Midi directly associates with actin, in vitro actin binding assays were performed. To further characterize the relationship between Midi and the acto-myosin ring, we tested the ability of actin to bind GST-Mid1 fragments with and without targeted mutations. Determining which members of the acto-myosin ring associate directly with actin, an essential ring component, will provide significant insight into the molecular details of cytokinesis. Research supported by NSF-Advance and GVSU.
Combinatorial Environmental Stress and Subtilisin-like Proprotein Convertase Expression. Soumya Pal, Rebekah Collins-Cronkright, Elizabeth Phinn, Patrick A. Wegman II, Sarah Regan, Tushar Ganjawala, Jeremy Lynd, and Joseph F. Sucic, University of Michigan--Flint, Biology Department
Subtilisin-like Proprotein Convertases (SPCs) are a family of eukaryotic endoproteases that function in the secretory pathway. Cleavage by SPCs activates a variety of proproteins, including hormones, growth factors, clotting factors, matrix-degrading metalloproteinases, receptors, and adhesion molecules. SPCs may be involved in mediating cellular responses to environmental stresses like heat shock and hypoxia. We used LoVo cells (human colon carcinoma) to examine the expression of the genes for four widely-expressed SPCs (furin, PACE4, PC5, and PC7) in response to conditions of heat shock (42[degrees]C), hypoxia (1% oxygen), and both heat and hypoxia. Control cells were incubated under standard cell culture conditions. RNA and protein were extracted from control cells and the cells under stress conditions at three time points (6 hours, 48 hours, and 96 hours of incubation under stress). Quantitative Real-Time PCR was used to evaluate SPC expression at the mRNA level, while western blotting was used to examine protein levels.
Regulation of the Mitotic-Activated Protein Kinase (MAPK) via Adenosine Deaminase (ADA) in the Developing Drosophila Eye. Cody Doyle, Melisa Babcock, Nathan Marzonie, and Gerard Paez, University of Michigan--Flint, Department of Biology
Adenosine deaminase (ADA) is a cytosolic enzyme that regulates the concentration of both intracellular and extracellular adenosine. It also modulates numerous physiological processes, including oxygen and metabolic balance in tissues, immune responses and signaling pathways. Adenosine receptors (AdoR) are members of the G protein-coupled receptor superfamily and mediate extracellular adenosine signaling, but the mechanism of adenosine signaling is still unclear. Adenosine deaminase can stimulate mitogenic and prosurvival actions by controlling Mitotic-Activated Protein Kinase (MAPK) family of kinases. The purpose of this project is a deeper understanding of the complexity of signal transduction pathways like Ras/MAPK regulated by Adenosine Deaminase. Our results suggests that AdoR is required for proper activation of Ras/MAPK pathway via ADA regulates to control the choice between cell division, cell growth, and cell differentiation in the Drosophila developing eye.
Regulation of p53 Expression by the RAS-MAP Kinase Pathway in Drosophila Melanogaster Eye. Sarhad Najor and Gerard Paez, University of Michigan--Flint, Department of Biology
Proper activation of the Ras/MAPK pathway is broadly required during development, and in many cases, signal transduction is unbranched. Thus, different mechanisms exist to properly regulate the large number of specific developmental outputs that are required by the activation of this pathway. In the developing eye, the phosphorytation of MAPK is itself another step in the regulation of cell growth and division in Drosophila melanogaster photoreceptor cells. We show that inhibition of MAPK phosphorylation, or pMAPK nuclear translocation, is sufficient to block cell growth, but not cell division. These data suggest that non-phosphorylated MAPK is sufficient to induce cell division, but not cell growth, once inside the nucleus of the cell. Here, we further refine the role of MAPK in Drosophila. We report evidence that suggests that activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We conclude that MAP kinase-dependent pathways help to regulate p53 levels by regulating the expression of p53 mRNA.
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|Date:||Sep 22, 2012|
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