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Identification and isolation of an azoreductase from Enterococcus faeciumMacwana, Susan Rebeka, January 2007 (has links) (PDF)
Thesis (M. S.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.
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Modeling the Effect of Cell Shape on GTPase Signaling in NeuronsRamirez, Samuel Andres January 2015 (has links)
<p>Biological processes such as cell division and synaptic plasticity are regulated by concentration gradients of signaling molecules. A number of biochemical mechanisms can result in intracellular signaling gradients. For example, restriction of diffusional flux of a chemical from one compartment to another will result in a transient gradient. A sustained gradient can be generated by opposite reactions such as phosphorylation and dephosphorylation of a signaling substrate taking place at different locations in the cell. More sophisticated mechanisms for non-uniform spatial signaling profiles include Turing type patterning and wave-pinning. It is becoming apparent that cell shape can regulate concentration gradients and modulate the downstream processes. In Chapter 1 we review how cell geometry can regulate intracellular signaling gradients in the context of the aforementioned gradient-generating mechanisms. The works reviewed make heavy use of mathematical modeling in order to investigate how reaction and diffusion taking place in complex cell geometries can modulate concentration gradients. That is a motivation for Chapter 2 where we implement a computational method to simulate reaction and diffusion on curved surfaces representing the cell membrane coupled with reaction and diffusion in the enclosed volume (representing the cell cytosol). To solve the reaction-diffusion equations on the surface we use the closest point method, a finite-difference technique that embeds the equations in the surrounding space. Such method is coupled with an embedded boundary technique to solve the equations in the enclosed volume with boundary conditions accounting for material exchange between surface and volume. The method is second-order convergent in the grid spacing despite a simple accuracy analysis predicts first-order errors. In Chapter 3 we use mathematical modeling in order to propose mechanisms accounting for the spatiotemporal dynamics of Rho-GTPase signaling at dendritic spines during synaptic plasticity. Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines (structural plasticity) through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation following synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Since Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased remains unclear. We model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine and interestingly, retention is enhanced by high diffusivity of Cdc42. These findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia.</p> / Dissertation
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New Congenital Mouse Model to Study Laminin Protein Therapy for Muscular DystrophyCoffey, Caroline B. M. 27 January 2016 (has links)
<p> Merosin deficient congenital muscular dystrophy type 1A (MDC1A) is caused by the loss of laminin-211 and laminin-221 heterotrimers which are most abundant in skeletal and cardiac muscle basal lamina; mutations in the LAMA2 gene cause the loss of these laminin isoforms. This absence of laminin-211/221 in MDC1A reduces the capacity for myofiber adhesion, loss of sarcolemmal integrity and subsequently the ability of the skeletal muscle syncytium to generate force in a coordinated and efficient manner. Patients experience progressive muscle wasting which confines them to a wheelchair at an early age and respiratory failure that leads to their untimely death. Currently, there is no effective treatment or cure for this devastating disease. Previous studies have shown that laminin-111, an embryonic form of laminin, delivered before disease onset can reduce muscle pathology and improve viability in the dyW-/- mouse model of MDC1A. These studies suggested that laminin-111 may act to strengthen and reinforce the sarcolemma and provide a protective niche for muscle repair. Since most patients are diagnosed with MDC1A after disease onset, we determined if laminin-111 could be beneficial after disease onset. Our studies suggest dyW-/- mice treated with laminin-111 after disease onset show improvement in muscle function and histology. Results from this study along with an understanding of laminin-111 pharmacokinetics will help pave the way in developing this protein as an exciting potential therapeutic for MDC1A patients. Duchenne Muscular Dystrophy (DMD) is the most common X-linked disease affecting 1 in 3,300 live male births. Patients with DMD suffer from severe, progressive muscle wasting and weakness with clinical symptoms first detected between 2 to 5 years of age; as the disease progresses patients are confined to a wheelchair in their teens and die in their early 20s mainly due to cardiopulmonary complications. DMD is caused by the loss of the sarcolemmal protein dystrophin (427kDa) due to mutations in the dystophin gene. When present, dystrophin acts as a scaffold linking the cell cytoskeleton to the extracellular matrix. This loss of dystrophin in DMD results in patients experiencing greater susceptibility to muscle damage via reduced structural and functional integrity of their muscle. One potential therapeutic avenue that needs to be explored involves increasing the levels of the ?7?1 integrin in order to compensate for the loss of dystrophin. To test this hypothesis, a muscle cell-based assay was developed in order to report ?7 integrin promoter activity with the intent of identifying molecules that promote ?7 integrin expression. Laminin-111 was identified as an enhancer of ?7 integrin expression. Theoretically, the identification of ?7 integrin enhancing compounds that help boost ?7?1 integrin expression as part of drug-based therapies may lead to a novel therapeutic approach for the treatment of this disease. Systemic laminin-111 treatment significantly reduces myofiber degeneration in both forms of MDC1A and DMD muscular dystrophy. This dissertation reinforces the potential of laminin-111 as a systemic protein therapy, capable of restoring sarcolemmal integrity thus reducing muscle disease progression. The importance of ?7 integrin in skeletal and cardiac muscle was highlighted here through the generation of the ?7-/-:: laminin-?2-/- double knockout mouse model. This mouse has never been studied before and could prove to be another important mouse model needed to explore therapeutic avenues for muscular dystrophy.</p>
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The evolution of protein folds from the perspective of structure motifsDybas, Joseph M. 18 December 2015 (has links)
<p> Understanding how protein structures evolve is essential for deciphering relationships between homologous proteins, which can inform structure classification and function annotation and aid in protein modeling and design methods. The observation that structure is more conserved than sequence, over the course of evolution, implies a model of evolution where sequences diverge within a discrete set of well-defined folds, which suggests that homology does not exist across fold definitions. However, as more structures have been experimentally solved and the coverage of the universe of folds has increased, the original view of a discrete fold space has been revised to include a more nuanced view of a continuous space defined by regions in which structural similarities can connect globally disparate topologies.</p><p> Structural, functional and evolutionary relationships are known to, in some cases, span fold definitions. A hallmark of relationships connecting disparate topologies is the conservation of local structure motifs within globally different folds. In order to systematically identify and analyze these relationships, a new approach to structure comparisons and structure classification is required. The goal of this work is to systematically identify evolutionary relationships between folds and to generate a classification of the fold universe that can accurately represent even the relationships that exist across disparate topologies. </p><p> An exhaustive library of supersecondary-structure motifs (Smotif), defined as two secondary structures connected by a loop, is established and characterized. A novel Smotif-based, superposition-independent structure comparison method (SmotifCOMP) is developed that quantitatively measures the Smotif-based similarity of compared structures in order to identify evolutionary relationships. SmotifCOMP is able to provide a quantitative and robust measure of similarity between disparate topologies since it does not rely on a global superposition. The comparison method is used to perform a systematic analysis of the SCOP Superfamilies and generate a non-hierarchical, network-based representation of the fold universe.</p><p> This thesis describes the development of a novel method of comparing structures and an improved representation of the relationships within the fold space. This work provides insight into the existence of evolutionary relationships between folds and strengthens the view of a connected and continuous fold universe.</p>
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Conformational and topographical studies of alpha-melanotropin "message" sequence and molecular modeling of the hMC1R melanocortin receptor.Haskell-Luevano, Carrie. January 1995 (has links)
The exciting and intriguing biological effects associated with amelanocyte stimulating hormone, α-MSH, have initiated a variety of studies to identify the biologically important conformations of the backbone and sidechains structures. To further investigate the topology of the critical "message" residues, several bicyclic (sidechain-to-sidechain) and x¹ restricted peptides have been synthesized and biologically examined, and selected analogues have been studied by NMR techniques and by theoretical molecular modeling studies. Bicyclic melanotropic peptides possessing the central sequence, Cys⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Trp⁹-Cys¹⁰-Lys¹¹, exhibited nearly identical nanomolar biological potencies in the lizard skin bioassay. Molecular modeling studies suggested the hypothesis of incomplete receptor binding by either the His or Trp residue as being responsible for the decrease in potencies relative to a-MSH, and the parent monocyclic peptides from which these bicyclic peptides were designed. This hypothesis is further supported by twodimensional NMR studies of a representative bicyclic peptide, Ac-Cys⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Trp⁹-Cys¹⁰-Lys¹¹-NH₂. Peptides synthesized to probe the topographical space of the tryptophan residue at position nine provided extraordinary results regarding the biological phenomena of prolongation. These peptides were based on the template, Ac-Nle⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Xaa⁹-Lys¹⁰-NH₂, where Xaa consists of the four isomers of β-MeTrp, DTrp , and L- or D-TCA residues. Some of these peptides were selected for studies on the cloned hMC1 melanocortin receptor. These studies resulted in a partial hypothesis accounting for the prolonged biological activities observed in other physiological assay systems. Conformational analysis by solution 20 NMR techniques revealed similar peptide backbone secondary structure features with main differences of structure occurring in the sidechain x¹ space. The implications and results are discussed. Homology-based molecular modeling of the hMC1 melanocortin receptor was also undertaken and provided evidence for ligand-receptor interactions which are being tested by receptor mutagenesis studies. This three-dimensional computer model provides an opportunity to probe detail chemical ligand-receptor interactions and further study differences in biological activities and biological mechanisms.
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Genetic Connectivity and Phenotypic Plasticity of Shallow and Mesophotic Coral Ecosystems in the Gulf of MexicoStudivan, Michael 12 June 2018 (has links)
<p> Coral reef ecosystems worldwide are facing increasing degradation due to disease, anthropogenic damage, and climate change, particularly in the Tropical Western Atlantic. Mesophotic coral ecosystems (MCEs) have been recently gaining attention through increased characterization as continuations of shallow reefs below traditional SCUBA depths (>30 m). As MCEs appear to be sheltered from many stressors affecting shallow reefs, MCEs may act as a coral refuge and provide larvae to nearby shallow reefs. The Deep Reef Refugia Hypothesis (DRRH) posits that shallow and mesophotic reefs may be genetically connected and that some coral species are equally compatible in both habitats. The research presented here addresses key questions that underlie this theory and advances our knowledge of coral connectivity and MCE ecology using the depth-generalist coral <i>Montastraea cavernosa</i>. Chapter 1 presents an overview of the DRRH, a description of MCEs in the Gulf of Mexico (GOM), and the framework of research questions within existing reef management infrastructure in the GOM. Through microsatellite genotyping, Chapter 2 identifies high connectivity among shallow and mesophotic reefs in the northwest GOM and evidence for relative isolation between depth zones in Belize and the southeast GOM. Historical migration and vertical connectivity models estimate Gulf-wide population panmixia. Chapter 3 focuses on population structure within the northwest GOM, identifying a lack of significant population structure. Dominant migration patterns estimate population panmixia, suggesting mesophotic populations currently considered for National Marine Sanctuary protection benefit the Flower Garden Banks. Chapter 4 quantifies the level of morphological variation between shallow and mesophotic <i>M. cavernosa</i>, revealing two distinct morphotypes possibly representing adaptive tradeoffs. Chapter 5 examines the transcriptomic mechanisms behind coral plasticity between depth zones, discovering a consistent response to mesophotic conditions across regions. Additionally, variable plasticity of mesophotic corals resulting from transplantation to shallow depths and potential differences in bleaching resilience between shallow and mesophotic corals are identified. The dissertation concludes with a synthesis of the results as they pertain to connectivity of shallow and mesophotic corals in the Gulf of Mexico and suggests future research that will aid in further understanding of MCE ecology and connectivity.</p><p>
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Neuroprotection during Acute Hyperthermic Stress| Role of the PKG Pathway in Neurons and Glia in the Protection of Neural Function in Drosophila melanogasterKrill, Jennifer 12 June 2018 (has links)
<p> The human brain functions within a narrow range of temperatures and variations outside of this range incur cellular damage and death and, ultimately, death of the organism. Other organisms, like the poikilotherm <i>Drosophila melanogaster</i>, have adapted mechanisms to maintain brain function over wide ranges in temperature and, if exposed to high temperatures where brain function is no longer supported, these animals enter a protective coma to promote survival of the organism once the acute temperature stress is alleviated. </p><p> This research characterized the role of different neuronal cell types, including glia, in the protection of brain function during acute hyperthermia, specifically looking at two protective pathways: the heat shock protein (HSP) pathway and the cGMP-dependent protein kinase G (PKG) pathway. Whole animal behavioral assays were used in combination with tissue-specific genetic manipulation of protective pathways to determine the specific cell types sufficient to confer protection of neuronal function during acute hyperthermia. Using the neuromuscular junction (NMJ) preparation, calcium imaging techniques were combined with pharmacological and genetic manipulations to test the hypothesis that alterations in ion channel conductance via endogenous mechanisms regulating the cellular response to high temperature stress alter neuronal function. </p><p> Expression of <i>foraging</i> RNAi to inhibit PKG expression in neurons or glia demonstrated protection of function during acute hyperthermia measured behaviorally through the extension of locomotor function. This extension of function with the tissue-specific inhibition of PKG was also confirmed at the cellular level using the genetically encoded calcium indicator (GECI), GCaMP3, to image calcium dynamics at the NMJ, where preparations expressing <i> foraging</i> RNAi could continue to elicit changes in calcium dynamics in response to stimulation. Over the course of this study, the mechanism underlying a novel glial calcium wave in the peripheral nervous system was characterized in order to elucidate glia’s role in the protection of neuronal function during acute hyperthermia.</p><p>
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Some properties of ovorubin, a caroteno-protein, from the eggs of Pomacea canaliculataNorden, Daphne Anne January 1962 (has links)
Ovorubin, apoprotein and ovorubin reconstituted from apoprotein and carotenoid were prepared from the eggs of Pomacea canaliculata australis, by adsorption on alumina CB or on carboxymethyl cellulose. Electrophoresis indicated an homogeneous preparation, but solubility tests indicated two components, very similar except for spectral differences. Paper chromatography revealed the presence of all the commonly occurring amino acids. 20% of the ovorubin molecule consisted of carbohydrate (glucosamine, galactose, mannose and fucose). The carotenoid component is in the trans form. Ovorubin was shown to possess a strong antitryptic activity, comparable with that of ovomucoid, which was not lost on removal of the carotenoid. The apoprotein was not more readily denatured than ovorubin, except after several precipitations with acetone. No direct correlation between changes in spectrum and inhibitory activity was demonstrated, neither was the spectrum modified by reaction with trypsin, Acetylation of amino groups of the apoprotein did not prevent inhibition, but did prevent recombination with the carotenoid. Ovorubin did not inhibit acetylated trypsin. A protein with antitryptic activity was isolated from eggs of Pila ovata gordoni, but was not extensively investigated. Trypsin slowly attacked ovorubin; pepsin produced a quite rapid fall in inhibition of trypsin and, after addition of trypsin and Chymotrypsin, the degradation product was isolated and shown to retain some antitryptic activity although the protein moiety was smaller. Ovorubin was shown to inhibit chymotrypsin and a protease from Aspergillus oryzae. Indications were obtained that an enzyme produced by B. subtilis was also inhibited. The nature of the second component in ovorubin preparations was discussed. The properties of ovorubin were compared with the proteins of hens' eggs; in composition and antitryptic action ovorubin is very similar to ovomucoid. A possible bacteriostatic role was suggested for ovorubin.
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The association of carotenoids with protein in certain invertebratesZagalsky, P. F. January 1964 (has links)
Crustacyanin, the blue carotenoprotein of the lobster carapace was obtained in a state homogeneous in cellulose acetate electrophoresis. It was crystallised and shown to be a globulin of large particle size, free from lipid and carbohydrate. The minimum molecular weight of the protein calculated on the carotenoid content, was found to be one-tenth of that expected from its size. Reversible changes in spectrum occurring on removal of salt were shown to be attended by dissociation into subunit of the size expected from the minimum molecular weight. Apocrustacyanin was shown to be of the subunit size and heterogeneous in cellulose acetate electrophoresis. Native cnustacyanin was reconstituted from the apoprotein and astaxanthin. Possible modes of binding of the carotenoid are suggested to account for the spectral changes occurring in urea. Electrophoretic similarities between crustacyanin treated with urea and apocrustacyanin are discussed in relation to the number of different subunits. It is suggested that crustacyanin is composed of a specific geometrical association of several small subunits, possibly different, and that the carotenoid stabilises the subunits in a configuration essential for the association and possibly also assisting in the binding. Ovoverdin, the green carotenoprotain of lobster ovary, was obtained homogeneous in cellulose acetate electrophoresis and shown to be a glycolipoprotein, precipitating at low ionic strength out soluble in distilled water. The possibility of two molecules of carotenoid being associated with each molecule of protein has been investigated. Astaxanthin-proteins were purified from the carapaces of Eriphia spinifrons, Carcinus maenas, Astacus astacus and Palinurus vulgaris. Their absorption spectra and properties arc compared with those of crustacyanin and ovoverdin. Glycolipoproteins containing a number of carotenoids have been purified from the ovaries of Carcinus maenas, Pecten maximus and Palinurus vulgaris, and from the eggs of Palinurns vulgaris. Their properties have been investigated and the ode of association of the carotenoids is discussed.
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Investigating the restraints upon recombinant protein production in mammalian cells via the manipulation of eEF2, eEF2K and mTORDean, Andrew January 2014 (has links)
Since their first clinical application in the 1980’s recombinant proteins have become an increasingly larger section of the drug market, growing into a multi-billion dollar global market. The pursuit of improving the design, production, and application of recombinant proteins for biotherapeutic uses is a key driver in industry and academia. The majority of the recombinant biotherapeutic proteins used in the clinic are produced in mammalian cell expression systems due to their capability to undertake human-like complex post translational modifications. The currently ‘gold standard’ mammalian cell expression system for the production of recombinant proteins is the Chinese hamster ovary (CHO) cell line. However, even with advances in mammalian cell expression technology, there is still a high cost and a long development period required for a recombinant protein therapeutic to go from design to market. As such, bottlenecks include the time taken for mammalian cells to grow and divide with slow doubling times compared to microbial systems and limited capacity to synthesise and secrete recombinant proteins. One of the cellular processes that underpins both cell growth and recombinant protein production is the translation of mRNA. Translation consists of three distinct steps: initiation, elongation and termination. One major cell signalling pathway that is considered a master regulator of both initiation and elongation of translation is mTOR, also involved in regulating ribosome biogenesis and cell proliferation. During the process of polypeptide elongation (mRNA translation), elongation factor 2 (eEF2) is a key control point that regulates protein synthesis via its de/phosphorylation. Phosphorylation of eEF2 results in its inactivation; slowing or halting elongation resulting in the attenuating of protein synthesis. This study set out to establish if manipulation of the mTOR signalling pathway and/or manipulation of phosphorylation of elongation factor 2 and the kinase that inactivates eEF2, eEF2K, in CHO cells impacts upon CHO cell growth and recombinant protein production yields. Transient expression of wild type and a Thr56Ala eEF2 mutant in CHOK1 cells affected the short term (24-48 hour) phosphorylation of eEF2 but did not appear to have an effect upon intracellular recombinant protein production and cellular growth in culture over 96 hours. Stable over expression of the wild type eEF2 construct in CHO cells resulted in a 2-fold increase in expression of eEF2 and a decrease in phosphorylation of eEF2 at the protein level; but the there was no change in the levels of total eEF2 mRNA expression. Stable expression of the Thr56Ala and Thr56Glu eEF2 mutants had a greater effect upon eEF2 expression resulting in a 3-5 fold increase in total eEF2 expression, however the phosphorylation of eEF2 was almost unchanged in an Ala56 eEF2 cell line, whereas it was reduced in the Glu56 eEF2 mutant cell 20 line. Growth of the CHO cells lines expressing the eEF2 mutants show that over expression of any of the eEF2 mutants resulted in a change in growth, but the Ala56 eEF2 mutant showed the largest change in cellular growth. Short term transient expression of recombinant firefly luciferase in the stable eEF2 cell lines revealed that the Thr56Ala mutant greatly increases the CHO cells total recombinant protein production. Further, mutation of eEF2 to Ala56 or Glu56 had little effect upon the mis-incorporation of amino acids during translation. The transient knockdown of eEF2K was achieved and this was shown to prevent eEF2 phosphorylation. However, CHO cells do not appear to tolerate the knockdown of eEF2K stably or at only very low levels. This suggests that a sustained, high level of eEF2K knockdown is lethal to the cell; which would result in the loss of eEF2 regulation for an extended period of time. Transient expression of the eEF2K shRNA into CHOK1D6 cells stably expressing firefly luciferase, a nonsecreted protein, resulted in a 5-fold increase in luciferase expression showing that knockdown of eEF2K increased the short term productivity of these cells. Interestingly, the study of CHO cell lines with varying recombinant monoclonal antibody protein production capacities revealed that levels of total and phosphorylated eEF2 did not appear to change in correlation with the mAb titre. These data suggest that eEF2 activity is tightly regulated across cell lines and is not directly related to recombinant protein secretion in these industrially used CHO cell lines The addition of 2 µM of PMA, an activator of mTOR signalling, increased antibody production in a low producer cell line but had no effect upon the antibody production from a high producing cell line. Together the data presented here shows that manipulation of eEF2 and eEF2K activity can enhance cellular growth and recombinant protein production from CHO cells. As such, new engineering approaches that allow the manipulation of both elongation and polypeptide synthesis combined with the secretory capacity of the cell are likely to yie ld new CHO host cells with more predictable recombinant protein capacity and further advance our understanding of the role of mRNA translation in controlling cell proliferation, and both global and recombinant protein synthesis.
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