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Sodium channels are required for cardiac cell-fate specification via a novel, non-electrogenic mechanism in zebrafish.Chopra, Sameer 06 December 2008 (has links)
Electrical signaling events are required for each human thought, feeling, and perception, the movement of our limbs, and the beat of our hearts. As the initiators of action potentials in excitable tissues, voltage-gated sodium channels play significant roles in both normal and pathological signaling events. In the heart, the opening of sodium channels initiates the cardiac cycle and mutations in the gene encoding the cardiac sodium channel are linked to heritable arrhythmias. Here we define a previously-unappreciated role for sodium channels in heart development. In zebrafish, knockdown of cardiac sodium channel expression in early embryos resulted in a failure of chamber morphogenesis and looping. We found these abnormalities to be associated with a significant deficit in the production of cardiac progenitor cells due to the perturbed expression of key transcription factors in early cardiac primordia. Pharmacological blockade of sodium channels did not recapitulate the effects of channel knockdown. These results suggest that voltage-gated sodium channels have evolved two distinct roles in the vertebrate heart: in addition to acting as the principal orchestrators of heart rhythm, they perform a previously-unappreciated, non-electrogenic function in early cardiogenesis. This dissertation describes the identification, cloning, and characterization of zebrafish cardiac sodium channel á and â subunits in early heart development and function.
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Towards a Unified Understanding of Eukaryotic Cell MotilityGruver, Jonathan Scott 16 February 2010 (has links)
Cell motility plays important roles in development, wound healing, and metastasis. Cells move either spontaneously, in a non-directed fashion, or in response to chemotactic signals, in a directed fashion. Even though they are often studied separately, both forms of motility share many complex processes at the molecular and subcellular scale, e.g., orchestrated cytoskeletal rearrangements and polarization. In addition, at the cellular level both types of motility include directionally persistent runs interspersed with reorientation pauses. The non-directed and chemotactic motility of Dictyostelium cells was studied quantitatively. Focusing first on chemotaxis, it was discovered that cells coordinate their speed and direction to increase chemotactic efficiency. Application of bimodal analysis, a method that compares time spent in persistent mode versus reorientation mode, to non-directed and chemotactic motility revealed that reorientation time is coupled with persistent time in an inverse correlation. Surprisingly, the inverse correlation holds for both non-directed and chemotactic motility so that the full range of Dictyostelium motility can be described by a single scaling relationship. Additionally, we found an identical scaling relationship for three human cell lines, indicating that the coupling of reorientation holds across species and making it possible to describe the complexity of cell motility in a surprisingly general and simple manner. We speculate that spontaneous cell polarity and the resultant directional persistence to be a fundamental aspect of cell motility that is quantitatively tuned by the presence of chemoattractant-induced signaling to increase chemotactic efficiency.
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MOLECULAR MECHANISMS OF ADAR2 LOCALIZATION AND SUBSTRATE SPECIFICITYXu, Ming 04 April 2006 (has links)
ADAR2-mediated adenosine-to-inosine (A-to-I) RNA editing can affect the coding potential, splicing pattern, stability, and localization of the targeted RNA transcripts. ADAR2 contains two double-stranded RNA binding motifs (dsRBM) and a conserved adenosine deaminase domain. To investigate how the dsRBMs of ADAR2 bind to natural substrates, we developed an NMR-based model of the complex formed between the two dsRBMs and an RNA duplex derived from a naturally-occurring ADAR2 substrate. These structural studies demonstrated that dsRBMs recognize specific structural determinants and hence contribute to substrate specificity. In addition, we demonstrated that the dsRBMs of ADAR2 differ in their ability to modulate subnuclear localization and editing activity although their sequences/structures are highly conserved, emphasizing the functional inequality between members of this conserved protein motif family.
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Non-visual arrestins bind mitogen activated protein kinases and regulate their signalingCoffa, Sergio 05 August 2011 (has links)
Arrestins are multifunctional signaling proteins, important for the regulation of signal transduction and the trafficking of G protein-coupled receptors (GPCRs). Recently, GPCR-arrestin interactions have been proposed to be necessary for activation of G-protein-independent signaling pathways, one of which is the activation of mitogen activated protein kinases (MAPKs). To investigate potential arrestin-MAPK interactions, we have used a variety of molecular tools including the co-expression of the individual domains of arrestin with single components of the c-Raf1-MEK1-ERK2 signaling cascade. We found that non-visual arrestins bind all three kinases, assembling c-Raf1, MEK1, and ERK2 along their short axis, with each kinase directly interacting with both domains.
To further investigate the interactions between arrestins and MAPK, we used alanine-scanning mutagenesis of residues on the non-receptor-binding surface of arrestin that are conserved between arrestin-2 and arrestin-3. We found that the substitution of arginine 307 with an alanine significantly reduced arrestin-2 binding to c-Raf1, whereas the interactions of this mutant with active phosphorylated receptors and the downstream kinases MEK1 and ERK2 were not affected. In contrast to wild type arrestin-2, Arg307Ala mutant failed to rescue arrestin-dependent ERK1/2 activation in arrestin-2/3 knockout MEFs. Interestingly, alanine substitution of the homologous arrestin-3 residue (lysine 308) did not significantly affect c-Raf1 binding or its ability to promote ERK1/2 activation. Together, these findings suggest that the two non-visual arrestins perform the same function via distinct molecular mechanisms. To further elucidate arrestin-MAPK interactions, we performed in vitro binding assays using pure proteins, and demonstrated that ERK2 directly binds free arrestin-2 and arrestin-3, as well as receptor-associated arrestin-1, arrestin-2, and arrestin-3. We have also shown that the arrestin-2 and arrestin-3 association with beta2-adrenergic receptors (β2ARs) significantly enhances ERK2 binding, yet has virtually no effect upon arrestins interactions with the upstream kinases c-Raf1 and MEK1.
Arrestins exist in three conformational states: free, receptor-bound, and microtubule (MT)-bound. Using conformationally biased arrestin mutants, we found that ERK2 prefers two conformations: MT-bound, mimicked by constitutively inactive arrestin-Δ7, and receptor-bound, mimicked by pre-activated arrestin-3A. Both mutants were able to rescue arrestin-mediated ERK1/2 activation in arrestin-2/3 double knockout fibroblasts. Lastly, we found that the arrestin-2 interaction with c-Raf1 is enhanced by receptor binding, whereas the interaction between arrestin-3 and c-Raf1 is not, thus suggesting that the two non-visual arrestins execute similar functions via diverse mechanisms.
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CA2+-SELECTIVE TRPM CHANNELS REGULATE IP3-DEPENDENT CA2+ OSCILLATIONS IN THE C. ELEGANS INTESTINEXing, Juan 01 December 2009 (has links)
PHARMACOLOGY
Ca2+-SELECTIVE TRPM CHANNELS REGULATE IP3-DEPENDENT Ca2+ OSCILLATIONS IN THE C. ELEGANS INTESTINE
Juan Xing
Dissertation under the direction of Professor Kevin Strange
Posterior body wall muscle contraction (pBoc) in the nematode Caenorhabditis elegans occurs rhythmically every 45¡V50 s and mediates defecation. pBoc is controlled by inositol-1,4,5-trisphosphate (IP3)¡Vdependent Ca2+ oscillations in the intestinal epithelial cells. The intestinal epithelium can be studied by patch clamp electrophysiology, Ca2+ imaging, genome-wide reverse genetic analysis, forward genetics, and molecular biology and thus provides a powerful model to develop an integrated systems level understanding of a nonexcitable cell oscillatory Ca2+ signaling pathway. Intestinal cells express an outwardly rectifying Ca2+ (ORCa) current (IORCa) with biophysical properties resembling those of TRPM channels. Two TRPM homologues, GON-2 and GTL-1, are expressed in the intestine. Using deletion and severe loss-of-function alleles of the gtl-1 and gon-2 genes, we demonstrate here that GON-2 and GTL-1 are both required for maintaining rhythmic pBoc and intestinal Ca2+ oscillations. Loss of GTL-l and GON-2 function inhibits IORCa 70% and 90%, respectively. IORCa is undetectable in gon-2;gtl-1 double mutant cells. These results demonstrate that (a) both gon-2 and gtl-1 are required for ORCa channel function, and (b) GON-2 and GTL-1 can function independently as ion channels, but that their functions in mediating IORCa are interdependent. IORCa, IGON-2, and IGTL-1 have nearly identical biophys+ical properties. Importantly, all three channels are at least 60-fold more permeable to Ca2+ than Na+.
Epistasis analysis suggests that GON-2 and GTL-1 function in a common signaling pathway with PLC× and IP3 receptors to regulate intestinal Ca2+ oscillations. PLC× via hydrolysis of PI(4,5)P2 (PIP2) regulates GON-2/GTL-1 function. Knockdown of PLC× by RNA interference (RNAi) inhibits channel activity ~80%. Inhibition is fully reversed by agents that deplete PIP2 levels. PIP2 added to the patch pipette has no effect on channel activity in PLC× RNAi cells. However, in control cells, 10 ÝM PIP2 inhibits whole cell current ~80%. Channel inhibition by phospholipids is selective for PIP2 with an IC50 value of 2.6 ÝM. Elevated PIP2 levels have no effect on channel voltage and Ca2+ sensitivity and likely inhibit by reducing channel open probability, single channel conductance and/or trafficking. We conclude that hydrolysis of PIP2 by PLC× functions in the activation of both the IP3 receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca2+ influx pathway. Calcium influx through GON-2/GTL-1 feedback regulates its activity and likely functions to modulate IP3 receptor function. PIP2-dependent regulation of GON-2/GTL-1 function may provide a mechanism to coordinate plasma membrane calcium influx with PLC£^ and IP3 receptor activity as well as intracellular Ca2+ store depletion.
Approved: ____________________________________ Date: ______________
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Caenorhabditis elegans as a model to study molecular mechanisms of methylmercury toxicityHelmcke, Kirsten Jeanne 18 January 2010 (has links)
Methylmercury (MeHg), a known neurotoxicant, is found in seafood, leading to regular exposure of humans to this compound. Many of the molecular targets and detoxifying components of MeHg toxicity, including glutathione (GSH), metallothioneins (MTs), and heat shock proteins (HSPs) have been identified; however, the molecular mechanisms of MeHg neurotoxicity are largely unknown. We used the model organism, Caenorhabditis elegans, to elucidate some of these mechanisms. We found that, although MeHg accumulated within C. elegans and caused a delay in development and a decrease in pharyngeal pumping rate, many endpoints, including life span, brood size, thrashing rate, and, most surprisingly, nervous system morphology, were unaffected. This finding led to the hypothesis that C. elegans has unique mechanisms for protecting its nervous system from MeHg neurotoxicity. We examined the involvement of GSH, MTs, and HSPs in MeHg toxicity in C. elegans and found that GSH levels were altered upon MeHg exposure, a glutathione s-transferase was highly upregulated upon exposure, and that the lack of MTs in MT knockout animals resulted in increased sensitivity to this toxicant. We also demonstrated that MeHg can induce hormesis in C. elegans, likely at least partially due to the involvement of GSTs in MeHg toxicity. As a result of our findings, we began to elucidate some of the molecular mechanisms of MeHg neuroprotection in the C. elegans model system. Our findings are highly valuable to the field of human health due to the regular exposure of humans to MeHg.
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Regulation of the CaMKIV/PP2A Signaling ModuleReece, Kelie M'liss 02 February 2010 (has links)
Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine kinase that plays a crucial role in the control of synaptic plasticity and T cell maturation. Activation of CaMKIV requires Ca2+/calmodulin binding and phosphorylation at T200 by CaMK kinase. Previous work from my laboratory showed that protein serine/threonine phosphatase 2A (PP2A) forms a complex with CaMKIV, and negatively regulates the phosphorylation state and activity of the kinase. My thesis studies focused on understanding the molecular mechanisms underlying the regulation and assembly of the CaMKIVPP2A complex. I demonstrate, using a novel CaMKIV phospho-specific antibody, that PP2A negatively regulates CaMKIV by directly dephosphorylating the kinase on its phospho-Thr200 residue within the kinase. In addition, I show that PP2A regulates the endogenous kinase in a tight and acute manner, but has little effect on the ectopically-expressed kinase. This differential regulation of endogenous versus ectopic CaMKIV by PP2A does not appear to be due to differences in the subcellular localization of the kinase, as both forms of the kinases exhibited similar subcellular distribution profiles. Rather, the differences of endogenous versus ectopic CaMKIV regulation by PP2A is due to the fact that the PP2A catalytic subunit (PP2Ac) binds more efficiently to the endogenous enzyme. However, overexpression of either the B or Bregulatory subunit of PP2A causes the recruitment of PP2Ac to the ectopic CaMKIV, thus facilitating the assembly of a CaMKIVPP2A complex. These B- and B-containing holoenzymes also preferentially dephosphorylate CaMKIV in vitro, thus indicating that they may be the primary modulators of CaMKIV in cells. Together, my findings provide new insights into the regulation of CaMKIV by an associated PP2A holoenzyme, and lay the foundation for future studies aimed at uncovering novel aspects of the biology of this key signaling module.
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Discovery, Optimization, and Understanding the Mechanism of Action of Small Molecules that Restore E-cadherin ExpressionStoops, Sydney Lear 09 April 2012 (has links)
E-cadherin is a transmembrane protein that maintains intercellular contacts and cellular polarity in epithelial tissues. The down-regulation of E-cadherin is thought to aid in the induction of an epithelial-to-mesenchymal (EMT) transition resulting in an increased potential for invasion into surrounding tissues and entry into the bloodstream. Loss of E-cadherin has been observed in a variety of human tumors resulting from somatic mutations, chromosomal deletions, proteolytic cleavage of E-cadherin, and most commonly silencing of the CDH1 gene promoter.
A novel High-throughput screen was developed to identify small molecules that restored E-cadherin expression in the SW620 cell line followed by medicinal chemistry employing iterative analog library synthesis to better identify the structure-activity relationship. Preliminary optimization of the screening hit has shown it is possible to synthesize small molecules that have an improved ability to restore E-cadherin expression compared to the initial screening hits.
Recent endeavors have been taken to elucidate the mechanism of action of these small molecules to restore E-cadherin expression. Quantitative PCR analysis has shown that E-cadherin mRNA expression occurs after 3 hours of treatment with active analogs, suggesting the small molecules are altering transcription of the CDH1 gene. This was supported by experiments conducted using various plasmid constructs containing truncated segments of the E-cadherin promoter region and luciferase reporter. It was shown that active analogs had a significant increase in luciferase activity as compared to DMSO or an inactive analog, which were used as controls. More specifically, we were able to narrow the site of action the active analogs to a 200 bp fragment of the E-cadherin promoter region.
Elucidation of the mechanism of action will aid in identifying the novel molecular target. Such information would allow for further development of more efficacious and potent small molecules as well as further research to understand the importance of this interaction in the role of EMT and as a therapeutic target.
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GABA synthesis in developing hippocampus: SNAT1 surfaces as a dynamic regulator of inhibitory synaptic transmissionBrown, Molly Nicole 10 April 2010 (has links)
GABA functions as the primary inhibitory neurotransmitter in the mammalian brain. In hippocampus, GABA serves multiple roles during development and throughout adulthood, which include: 1) orchestrate synapse maturation and synaptogenesis, 2)
maintain inhibitory synaptic transmission, and 3) regulate the excitability and synchronize the neuronal output of principal neurons. Because normal brain function requires intact inhibitory synaptic transmission, interneurons must possess mechanisms to adapt synaptic GABA during a wide range of activity states. I hypothesized that one effective mechanism may involve regulating GABA synthesis and vesicular GABA content through modulating the supply of glutamine, an indirect GABAergic metabolic precursor, by System A glutamine transporters. In addition, because GABA serves critical roles during early hippocampal development we hypothesized that the role of
System A transporters at inhibitory synapses also may be developmentlally regulated. Therefore, this dissertation explored if modulation of System A surface activity dynamically regulated GABA synthesis and inhibitory synaptic transmission in response to changing developmental and neuronal activity states. Using electrophysiology in hippocampal slices in conjunction with protein expression studies and uptake assays in synaptosomes, I demonstrate that System A transporters serve both a constitutive and activity-dependent role in modulating vesicular GABA content and inhibitory synaptic strength. Synaptic depolarization up-regulates the surface activity of System A transporters and thus induces an increase in vesicular GABA content in both immature and mature hippocampus. However, because System As basal uptake activity and therefore its constitutive contribution to vesicular GABA content diminishes over the first two postnatal weeks, its role in mature hippocampus is only manifest in an activity-dependent manner. Furthermore, my results support that these constitutive and activitydependent roles are likely mediated by the SNAT1 subtype of System A transporters. Therefore, my findings strongly support the hypothesis that the surface activity of SNAT1, regulated both by depolarization and by developmental cues, is the key component in a novel mechanism to dynamically link metabolic demand for GABA with vesicular GABA content and inhibitory synaptic strength.
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Cardiovascular and Neuropsychiatric Consequences of a Genetic Loss of the High-Affinity Choline Transporter (CHT)English, Brett Alan 12 April 2010 (has links)
Acetylcholine (ACh) was one of the first neurotransmitters discovered and has been implicated in regulating a number of physiologic processes within the CNS and the periphery. Cholinergically-mediated physiology requires continuous turnover of ACh by the biosynthesis machinery to maintain cholinergic tone. High-affinity choline uptake (HACU), mediated by CHT in cholinergic terminals is pivotal for efficient ACh production and release. Cardiovascular function relies on a balanced integration of noradrenergic and cholinergic innervation of the heart. The cardiovascular impact of diminished CHT expression has not been directly examined, due to the transporters inaccessibility in vivo. We describe findings from cardiovascular studies using transgenic mice that bear a CHT genetic deficiency. Whereas CHT knockout (-/-) exhibit early postnatal lethality, heterozygous (CHT+/-) mice survive, exhibiting normal spontaneous behaviors. However, the CHT+/- mouse heart displays significantly reduced levels of HACU, accompanied by reduced levels of ACh. Telemeterized recordings of cardiovascular function in these mice reveal basal tachycardia and hypertension. After treadmill exercise, CHT+/- mice exhibit slower heart rate recovery, consistent with diminished cholinergic reserve, a contention validated through direct vagal nerve stimulation. Functional studies revealed an age-dependent decrease in fractional shortening and increased ventricular fibrosis, consistent with progressive ventricular dysfunction. Lastly, we show that the hypomorphic allele (Ile89Val) in human CHT is associated with overall symptom severity in patients with major depressive disorder (MDD) and was shown to be selectively overtransmitted in the combined subtype of attention-deficit hyperactivity disorder (ADHD). The identification of cardiovascular phenotypes in mice with deficits in CHT may provide potential biomarkers for the identification of autonomic dysfunction in a number of cardiovascular and neuropsychiatric disorders. Autonomic dysfunction has been indentified in a number of neuropsychiatric disorders contributing to morbidity and mortality. The increased allele frequency and selective transmission of the hypomorphic CHT allele in patients with psychiatric disorders demonstrates the importance of CHT not only in regulating CNS processes, but in regulating autonomic processes in patients with these disorders. Further studies examining the role of CHT in regulating autonomic imbalance may provide potential targets for novel therapeutics.
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