Browsing by Subject "Biophysics"
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Item Molecular Cloning, Expression, and Regulation of the Na+/Myo-Inosiotl Cotransporter Gene(1996-08-01) Zhou, Cheng; Chaitin, Michael; Easom, Richard; Garner, MargaretZhou, Cheng, Molecular Cloning, Expression, and Regulation of the NA+/Myo-Inositol Cotransporter Gene. Doctor of Philosophy (Biomedical Sciences), August 1996. Mammalian cells respond to osmotic stress by accumulation of high concentrations of intracellular osmolytes. Osmotic-induced accumulation of the osmolyte, myo-inositol (MI), is achieved through activation of the NA+/MI cotransporter. Hypertonic stress results in elevated NA+/MI cotransporter mRNA abundance and transcription rate, and increased transporter activity. The goals of this dissertation are to establish the osmoregulation of the NA+/MI cotransporter gene expression in lens cells, and to investigate the transcriptional regulation of the NA+/MI cotransporter gene. Expression of the Na+/MI cotransporter in cultured bovine lens epithelial cells (BLECs) was demonstrated by RT-PCR amplification and Northern blot analysis. Hypertonic stress resulted in induction of the NA+/MI contransporter mRNA abundance in cultured BLECs. The induction patterns of the NA+/MI cotransporter and aldose reductase mRNA abundance by hypertonic stress indicated that osmoregulation of MI and sorbitol accumulations were regulated in concert. Accumulation of MI is an early-onset protective system, which is suppressed by the elevated sorbitol, the late-onset protective system. 5’-RACE analysis indicated that multiple transcription start sites were utilized in controlling of the expression of the NA+/MI cotransporter. Osmotic stress resulted in preferential utilization of a hypertonic promoter a. The bovine NA+/MI cotransporter gene was cloned and analyzed. The regulation of the Na+/MI cotransporter expression was investigated by transient transfection assays using promoter-luciferase constructs. Although multiple promoters were functional in cultured BLECs, only the hypertonic promoter a was osmotically responsive. Characterization of this osmotic-responsive element(s) between -536 to -300 bp upstream of the hypertonic transcription start site a. The studies presented in this dissertation refined the osmoregulation of the Na+/MI cotransporter gene expression. Hypertonicity induces MI accumulation by activation of an osmotic-responsive promoter. The consequences of the activation of this promoter lead to more cotransporter mRNA, more cotransporter protein, and higher transporter activity, resulting in accumulation of a higher concentration of intracellular Mi.Item Psalmotoxin-1 and nonproton ligand interactions with acid-sensing ion channels(2015-05-01) Smith, Rachel N.; Gonzales, Eric B.; Dillon, Glenn H.; Sumien, NathalieAcid-sensing ion channels (ASICs) are trimeric, sodium-selective channels activated by extracellular protons. Although ASICs are intriguing molecular targets for pharmacological agents, there remains a lack of selective compounds that differentiate ASIC subtypes. The peripherally located ASIC3 activates with the simple removal of calcium. Additionally, nonproton ligands, like 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate ASIC3 via the nonproton ligand sensor domain (NPLSD). A pair of glutamates in rat ASIC3 (E79 and E423) responsible for GMQ activation is present in ASIC1, despite having no direct modulation effect on the channel. We proposed that nonproton ligand activation of ASIC1 may be state dependent, and relies on expansion of the NPLSD in order for GMQ to reach the binding site and exert its effects. We utilized two features of ASICs in order to test our hypothesis with whole cell and outside out patch-clamp electrophysiology. First, we induced a persistent current in chicken ASIC1 (cASIC1) via a natural venom toxin, Psalmotoxin-1 (PcTx1). We determined that GMQ acts as a molecular wedge by prying apart the transmembrane domains of the cASIC1-PcTx1 protein complex and potentiating ASIC-current. This led us to better understand that the NPLSD is intact in cASIC1 and is sensitive to GMQ additions, albeit in a state-dependent manner. We then theorized that direct activation of rASIC3 by GMQ is possible due the channel’s interaction with extracellular calcium, and were interested in introducing feature into the cASIC1 channel. We generated a chimeric ASIC combining the extracellular, transmembrane, and intracellular domains of rASIC3 and cASIC1 in order to individually isolate the calcium and nonproton ligand effects on channel activation. This chimera, termed cASIC1 (rASIC3-TM/ITC), is comprised of the extracellular domain of cASIC1 and the transmembrane/intracellular domains of rASIC3, and can be activated by GMQ in the absence of calcium similarly to wild-type rASIC3. Thus, GMQ activation was introduced in cASIC1 by replacing the transmembrane domains with those of ASIC3 suggesting that the ASIC3 TM domains dictate NPLSD influence on channel activity. Taken together, we identified that channel state influences nonproton ligand interaction with ASICs, and the transmembrane domains are critical for this interaction.Item Study of Cross Bridge Kinetics in Hypertrophic Ventricular Muscle(2009-05-01) Muthu, Priya; Borejdo, JulianCardiovascular diseases are the leading cause of mortality worldwide; with heart failure being highly prevalent in most affluent parts of the world. There is a need for a better understanding of the mechanism underlying these diseases. Familial hypertrophic cardiomyopathy (FHC), one such disease, is a genetic disorder of the heart characterized by increased growth or hypertrophy in the thickness of the wall of the left ventricle, the largest of the four chambers of the heart. This research project is focused on one kind of FHC, the D166V mutation in the regulatory light chain in myosin, which is associated with a particularly malignant form of the disease. The overall goal of this project was to study cross bridge kinetics (contraction and ATP utilization) in cardiac muscle from transgenic mice and to develop assays to apply this to human samples. The real time orientation changes of myosin and actin during a single cross bridge cycle beginning in a state of rigor was studied by Fluorescence anisotropy. Rabbit psoas fibers were isolated and used to achieve imaging of a few fluorophores or cross bridges. This technique was then applied to study cardiac myofibrils from transgenic mice, carrying the mutation causing the disease (FHC). Methods to achieve single molecule detection to aid studying human samples suffering from this disease were developed using silver island films, monolayers of nanoparticles and surface plasmon coupled emission. The conclusions of this dissertation were that a mutation in a light chain in myosin cause changes in the cross bridge kinetics. Myofibrils from the mutated mice displayed a significant slower rate of detachment during contraction as well as increased ATPase activity, which if severe enough could cause the heart to compensate by increasing wall thickness (hypertrophy). Despite significant clinical advances in the treatment of various cardiovascular diseases, mortality rates remain high. No therapy currently exists to treat or delay progression from hypertrophy to heart failure. This proposal help answer an important question regarding the molecular basis of FHC-mediated pathology in the heart. Also, achieving imaging of a single fluorophore has numerous implications in the biological field, like studying ligand-receptor interactions in live cells, involvement of protein molecules in internalization of bacteria by cells, monitoring the conformational fluctuations of DNA, diagnosis of prion diseases and also in detection of viruses at an early phase of infection.