Browsing by Subject "Signal transduction"
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Item Circadian Regulation of L-Type Voltage-Gated Calcium Channels in Avian Retina(2014-11-25) Huang, Chia-YuThe circadian clock is an endogenous time-keeping mechanism that allows an organism to synchronize itself with external time cues and prepares the organism to anticipate upcoming environmental changes on a daily basis. The retina is a light-sensitive neuronal tissue located in the back of the eye. The circadian clocks in the retina enable the retina to anticipate daily ambient illumination over at least twelve orders of magnitude and initiate the adaptive processes with visual system throughout the course of a day. The retinal photoreceptors are responsible for phototransduction and transmitting the visual information into the brain. Unlike most neurons, photoreceptors do not fire action potentials, and they release neurotransmitter in a sustained manner, which is governed by the L-type voltage-gated calcium channels (L-VGCCs). The mRNA and protein expression of the ?1 pore forming subunit of L-VGCCs are under circadian control, in which the protein expression of L-VGCC?1 with a corresponding increase in the L-VGCC current density is higher at night than during the day. Using the chicken embryo as a model system, an integrative strategy was used through combining biochemical, molecular, morphological, and electrophysiological analyses to investigate cellular mechanisms of the circadian regulation of L-VGCCs in the photoreceptors. Three important cell signaling molecules and their pathways were investigated in this dissertation: calcineurin, mechanistic/mammalian target of rapamycin complex 1 (mTORC1), and AMP-activated protein kinase (AMPK). The activities of the protein phosphatase calcineurin, as well as the protein kinase mTORC1 exhibited circadian oscillation with their activities higher at night than during the day, while the activities of AMPK are greater during the day compared to the activities at night. Inhibition of calcineurin and mTORC1 dampened the current densities and protein expression of L-VGCCs at night, while activation of AMPK decreased L-VGCC currents at night. These signaling molecules interacted with cAMP-Ras-MAPK and cAMP-Ras-PI3K-AKT signaling pathways to modulate the L-VGCC trafficking from the cytosol onto the plasma membrane in a circadian phase-dependent manner. The results demonstrated that the complex of cellular signaling pathways participated in the circadian regulation of L-VGCCs in the photoreceptors. Understanding the molecular mechanism underlying the circadian regulation of L-VGCCs in cone photoreceptors will provide important knowledge on how circadian clocks regulate retinal physiology and function in healthy states. Changes in L-VGCCs and these cell-signaling molecules might be indicators or biomarkers for age-related macular degeneration or other retinal degenerative diseases.Item Design and evolution of synthetic biological systems(2006-08) Tabor, Jeffrey Jay; Ellington, Andrew D.The study of biology has undergone a fundamental change due to advancements in genetic engineering, DNA synthesis and DNA sequencing technologies. As opposed to the traditional dissective mentality of discovering genes via genetics, describing genetic behaviors through biochemistry, and then drawing diagrams of functional networks, researchers now have the potential (albeit limited) to construct novel biological molecules, networks, and even whole organisms with user-defined specifications. We have engineered novel catalytic DNAs (deoxyribozymes) with the ability to 'read' an input DNA sequence and then 'write' (by ligation) a separate DNA sequence which can in turn be detected sensitively. In addition, the deoxyribozymes can read unnatural (synthetic) nucleotides and write natural sequence information. Such simple nanomachines could find use in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of difficult to detect (short) nucleic acids such as microRNAs. As an extension of in vitro biological engineering efforts, we aimed to construct novel signal transduction systems in vivo. To this end, we used directed evolution to generate a catalytic RNA (ribozyme) capable of creating genetic memory in E. coli. In the end we evolved an RNA which satisfied the conditions of our genetic screen. Rather than maintaining genetic memory, however, the RNA increased relative cellular gene expression by minimizing the translational burden it imposed on the host cell. Interestingly, detailed mutational analysis of the evolved RNA led us to new studies on the relationship between ribosome availability and stochasticity in cellular gene expression, an effect that had frequently been alluded to in the literature, yet never examined. We have also taken a more canonical approach to the forward engineering of biological systems with unnatural behaviors. To this end, we designed a protein-based synthetic genetic circuit that allows a community of E. coli to function as biological film, capable of capturing and recapitulating a projected light pattern at high resolution (theoretically 100 mexapixels). The ability to control bacterial gene expression at high resolution could be used to ‘print’ complex bio-materials or deconvolute signaling pathways through precise spatial and temporal control of regulatory states.Item Human glutamate transport: Aspects of regulation(Texas Tech University, 1997-08) Ganel, RaquelliGlutamate is the major excitatory neurotransmitter in the CNS including the retina. Termination of synaptic glutamate transmission and prevention of excitotoxicity depend on rapid removal of glutamate by neuronal and glial high affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least four subtypes of sodium-dependent mammalian glutamate transporters exist. The following studies demonstrate that Y-79 human retinoblastoma cells express a single sodium-dependent glutamate uptake system with a Km of 1.7 ± 0.42 |LIM, that is inhibited by the glutamate uptake inhibitors dihydrokainate and DL-Threo-B-hydroxyaspartate. The protein kinase C activator phorbol 12- myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC50 = 0.56 ± 0.05 nM). This inhibition resulted from a 5-fold decrease in the transporter's affinity for glutamate, without significantly altering its maximal velocity, and was blocked by pre treatment with the kinase inhibitor staurosporine. The neuropeptide substance P produced a concentration-dependent inhibition of glutamate uptake (IC50 = 0.46 ± 0.14 nM). This inhibition is not mediated via any of the known tachykinin receptors. A strong inhibitory effect of glutamate uptake was also caused by the 25-35 amino acid fragment of beta-amyloid protein, the main component of the senile plaques found in the CNS of patients with Alzheimer's disease. The antagonist SR 142801 blocked both substance P's and beta-amyloid's inhibitory effects, suggesting that both peptides inhibit glutamate uptake via activation of the same site. A kinetic study shows that the inhibition of glutamate uptake caused by substance P resulted from a 75% reduction in the transporter's maximal velocity, without significantly altering its affinity for glutamate, indicating a mechanism other than protein kinase C activation. These studies provide evidence for modulation of glutamate uptake by substance P via a novel site not coupled to the activation of protein kinase C. Beside having a physiological role in modulation of glutamatergic neurotransmission, this site may be involved in the pathophysiology of beta-amyloid protein.Item Regulation of calcium stores in normal and diabetic endothelial cells(Texas Tech University, 2000-12) Sanka, Shankar ChittaranjanCytosolic Ca^^ ([Ca^^]*'^^) mediates many cellular ftinctions, e.g.. cell growth, motility, secretion, etc. In many cell types, ion transport processes appear to be dependent on metabolism of glucose for maximal activity. In certain cell types, a strict coupling between glycolysis and the acfivity of Endoplasmic Reticulum Ca^"-ATPases (SERCA). involved in regulating Ca^^ homeostasis, has been suggested. In diabetes, glucose homeostasis is altered. We hypothesize that Ca^^ homeostasis in microvascular endothelial cells from diabetic animals is altered due to a dysfunction of glycolysis coupling the activity of SERCA. We further hypothesize that endosomal/lysosomal (E/L) compartments exhibiting SERCA are involved in this dysfunction. Our data indicated that agonist stimulation (ATP, vasopressin, angiotensin-II)elicited [Ca^"]^^* increases (independent of extracellular Ca^^) that were larger in endothelial cells from diabetic than from normal animals. Simultaneous measurements of [Ca^^]'^^' and Ca^^ in E/L compartments ([Ca^^]^) using fluorescence spectroscopy, indicated that E/L compartments released Ca^^ following agonist-stimulation. The magnitude of the Ca'* release was significantly larger in microvascular endothelial cells from diabetic rats. SERCA inhibitors elicited Ca^^ releases from E/L compartments in both normal and diabetic models. The magnitude of the [Ca^^]^ release was however similar among normal and diabetic cells. Immunocytochemical experiments demonstrated that 60% of E/L compartments exhibited SERCA. These data indicate that (a) E/L compartments are important for Ca^^ homeostasis in microvascular endothelial cells from both normal and diabetic models; (b) Ca^^ regulation in E/L compartments is different in cells from a diabefic model, (c) the compartment involved in altered Ca'* homeostasis in diabetes is unknown.Item The C-Terminus of Transmembrane Helix 2 (TM2) of the Escherichia coli Tar Chemorecptor Determines Signal Output and Ligand Sensitivity(2012-11-20) Adase, Christopher A. 1981-Methyl-accepting chemotaxis proteins MCPs can bind one or more receptor- specific ligands. In the case of the Tar MCP of Escherichia coli (TarEc), a primary attractant ligand is aspartate. Its binding to the periplasmic domain of Tar generates a conformational change that is transmitted via helix 4 transmembrane helix 2 (TM2). An inward movement of TM2 initiates a transmembrane signal to the cytoplasmic HAMP (histidine kinases, adenyl cyclases, methyl-accepting proteins, phosphatases) domain. Baseline CheA kinase-stimulating activity and ligand-induced responses are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). The cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in TarEc, is of particular importance. These residues are not highly conserved among transmembrane receptors having a HAMP domain, although there are almost always some aromatic residues in this region. The question thus becomes what properties of this aromatic anchor are necessary for proper signal transduction. In this dissertation, I studied the effect on TarEc function by substituting all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. This library of TarEc variants allowed the direct assessment of the effect of the residue composition of the aromatic anchor and led to a model of how the wild-type anchor maintains the base-line signaling state in TarEc. Additional receptor variants containing double aromatic tandems and Ala substitutions for the periplasmic Trp residue were created, and the aromatic residues were also shifted in position within the six residues 207-212. Trp, Tyr, and Phe, in that order, had the greatest effect on function when they were moved to novel positions. It was also discovered that Gly-211 plays a critical role in maintaining receptor function. A model was generated that proposes that Gly-211 plays a role in maintaining the flexibility of the TM2-HAMP domain connector. The results suggest that the signaling properties of the transmembrane sensor kinases of two-component systems can be predicted by the nature of their TM2-HAMP connections. It may also be possible to modulate their activity in a controlled way by manipulating the amino acid sequences that comprise those connections.Item The contribution of quorum sensing to the pathogenesis of pseudomonas aeruginosa in burn wound infections(Texas Tech University, 2001-05) Rumbaugh, Kendra P.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes severe infections in burned patients. In this work, we examined the contribution ofthe cell-to-cell communication systems or quorum sensing (QS) systems to the pathogenesis of P aeruginosa infection of burn wounds. For these studies, we used the thermally-injured mouse model and specific mutants that carry deletions in genes encoding specific components of the P aeruginosa QS systems {lasR, rhlR, lasl and rhll). In comparison with their parent strain (PAOl), all mutants displayed decreased lethality. The mutants were defective in their ability to spread systemically throughout the body of the mice. In addition, the lasR (PAO-Rl) and the lasl/rhll (PAO-JP2) mutants were defective in their ability to spread locally within the burned skin at 8 and 16 hours post-bum/infection. The defects in the PAO-JP2 strain were complemented upon the introduction of a plasmid carrying intact lasl and rhll genes. To determine if the defect in PAO-JP2 is due to the loss of one or more of the QScontrolled virulence factors, isogenic mutants that carry deletions in lasA, lasB, lasAAasB, toxA, lasB/toxA or rpoS were examined. With the exception of the rpoS mutant, all mutants were defective in their in vivo virulence. However, none was as severely defective as PAO-JP2. Our attempt to ascertain the role of autoinducers as virulence factors was confounded by the influence of the solvent used to purify them. Host production of cytokines in response to P aeruginosa infection in bum wounds was examined using the Multi-Probe Template/RNase protection assay. The expression of several proinflammatory and hematopoietic cytokines was up-regulated in burned mice infected with PAOl at 40 hours post-bum/infection. In contrast, the expression of most of these cytokines was not enhanced in burned mice infected with PAO-JP2. These results suggest that: (1) the QS systems play an important role in the pathogenesis of P aeruginosa in bum wound infections; (2) their effects may be contributed to by autoinducers or other, yet undefined, QS-controlled factors; and (3) QS may play a role in modulating the host immune system in response to P. aeruginosa.