Browsing by Subject "fluorescence"
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Item Effect of Phosphorylation on Muscle Physiology and Biophysical Characterization of Mutations Responsible for Familial Hypertrophic Cardiomyopathy(2016-08-01) Duggal, Divya; Borejdo, Julian; Gryczynski, Ignacy; Clark, Abbot F.Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in the pathogenesis of this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E and Troponin I mutation, R21C. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to 20. Autofluorescence and photobleaching were minimized by using a relatively long-lived red-emitting dye. In case of K104E, mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. In case of R21C, differences were investigated in the left (LV) and right ventricle (RV) mutant where it was found that the mutation imposed significant difference in the distribution of angles that actin makes with thin filament axis: during contraction, actin angles from LV were more tightly distributed compared to actin angles from RV. Collectively, the data indicates that the mutation-induced changes in the interaction of myosin with actin during the contraction- relaxation cycle may contribute to altered contractility and the development of FHC. Phenotypic differences of the R21C mutation in the left versus right mouse ventricles, even though both ventricles express the same isoform of the cardiac highlights the importance of functional differences between the two ventricles of cardiac disease.Item OPTIMIZATION OF TWO METHODS FOR ASSESSING CELL VIABILITY AND CYTOPROTECTION IN C6 ASTROCYTES(2014-03) Kubelka, Nicholas K.; Rybalchenko, Nataliya; Singh, MeharvanPurpose: The purpose of this study was to determine the best method for measuring cell viability in the rat C6 astrocyte cell model in response to two cytotoxic insults, hydrogen peroxide (H2O2) and iodoacetic acid (IAA). Methods: Two assays were evaluated: calcein-AM assay for detecting live cell number, and a flow cytometry-based assay to assess live versus dead populations. Cells were treated with 0, 10, 20 or 50 μM H2O2 (2 hours) or IAA (3 hours). The calcein-AM assay was evaluated in a 96-well plate format. Flow cytometry results were obtained using a C6 Accuri model Flow Cytometer, and cells were stained with both calcein-AM (peak emission range 485-535 nm, fluorescence channel 1 (FL1)) and ethidium homodimer (peak emission range 610-30 nm, fluorescence channel 3 (FL3)). Results: Treated cells displayed a concentration dependent decrease in cell viability (calculated EC50 = 18μM for H2O2, 23 μM for IAA). Cells treated for 3 hours with IAA showed a concentration dependent transition from high FL1, low FL3 fluorescence to low FL1, high FL3 fluorescence, indicating a transition from high to low viability. Conclusions: This study describes two methods for cell viability detection: calcein fluorescence by high-throughput analysis and simultaneous calcein and ethidium homodimer staining for flow cytometry cell gating for individual cell analysis. Having successfully utilized both the 96-well assay and the flow cytometry protocols to assess cell viability, we plan to extend the current studies by assessing how brain-active steroids protect glia from insults relevant to brain aging and certain neurodegenerative diseases.Item Study of Spatiotemporal Orientation and Cross Bridge Kinetics in Ventricular and Skeletal Muscles(2016-05-01) Nagwekar, Janhavi; Borejdo, Julian; Hodge, Lisa M.; Gryczynski, Ignacy