Browsing by Subject "Biochemistry"
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Item Analysis of an Arabidopsis mutant with altered stress responses(Texas Tech University, 2003-12) Shen, YinPlants face a variety of conditions that cause biotic and abiotic stress. Since plants are sessile, and therefore cannot move to escape stressful conditions, they have evolved complex strategies to survive harsh environments. One such strategy is the ability to quickly alter the expression of genes, which, in effect, acclimates the plant. Glutathione S-transferases (GST) are an example of these genes that are induced under stressful conditions. In order to identify the mechanisms that regulate stress responsive gene expression, I used a genetic strategy to identify stress signaling mutants in which the luciferase (LUC) reporter was expressed under control of the stressresponsive Arabidopsis GST6 promoter. After chemical mutagenesis by ethyl methanesulfonate (EMS), a number of potential mutants that affect the expression of the GST6:LUC reporter gene, and presumably, the stress signaling pathways were isolated. One of the mutants, cdg6 (constitutively down-regulated GST6), is dominant and has altered responses to abscisic acid (ABA), ethylene, and salicylic acid (SA). In addition, physiological studies of cdg6 mutant plants showed that the gene is not involved in the ethylene signaling or synthesis pathways. A transcriptional profile of cdg6 using microarray analysis gave us a genome-wide view of gene expression alterations in mutant plants compared to the wild type plants. This analysis showed that endogenous GST genes are down regulated in cdg6, while a number of defense related genes such as chitinase were up regulated. These data indicate that cdg6 may represent a mutation in a gene that regulates defense responses.Item Application of enzymatic catalysis and galvanic processes for biosensor development(2011-08) Zaccheo, Brian Andrew; Crooks, Richard M. (Richard McConnell); Browning, Karen; Hoffman, David; Johnston, Keith P.; Stevenson, KeithMethods for integrating enzyme systems with electrochemical reactions having applications to diagnostic sensing are described. Diagnostic tests that include biological molecules can be classified as biosensors. Existing testing methods often require trained technicians to perform, and laboratory settings with complex infrastructure. The theme of this dissertation is the development of methods that are faster, easier to use, and more applicable for non-laboratory environments. These goals are accomplished in systems using enzymatic catalysis and galvanic processes. Two biosensors with specific model pathologies have been designed and demonstrated in this study. The first assay senses a DNA fragment representing the Epstein Barr virus and uses enzyme-mediated Ag deposition over a v microfabricated chip. The chip contains a specially designed pair of electrodes in an interdigitated array (IDA). Detection is signaled by a change in the resistance between the two electrodes. The second biosensor discussed in this study is targeted towards the digestive enzyme trypsin. It is selfpowered due to its construction within an open-circuit galvanic cell. In this system, a small volume of blood serum is introduced onto the device over barriers made of protein and Al that block the anode from solution. In the presence of trypsin, the protein gel is rendered more permeable to sodium hydroxide. Adding hydroxide initiates the dissolution of the Al layer, closing the cell circuit and illuminating a light-emitting diode (LED). A relationship was observed between LED illumination time and trypsin concentration. Biosensors that utilize enzymes to generate or amplify a detectable signal are widely used, and the final project of this study uses a nanoparticle based approach to protect the catalytic activity of alkaline phosphatase (AlkP) from hostile chemicals. By incubating Au colloid with AlkP overnight and adding Ag+, core@shell nanoparticles of Au@Ag2O can be isolated that show AlkP activity. The resulting enzyme-metal composite material was analytically characterized and demonstrated greater activity in the presence of organic inhibitors relative to either wild type vi or Au colloid-associated AlkP without the Ag2O shell. The stabilization procedure is complete in one day using a onepot synthesis. This method may provide opportunities to carry out biosensing chemistry in previously incompatible chemical environments.Item Breed differences in seminal plasma chemistry; Implications for(2012-12) Yeomans, Glenn; Prien, Samuel D.; Thompson, Leslie D.; Ballou, Michael A.Seminal plasma serves as a nutrient rich medium for spermatozoa to function and survive. Several components of seminal plasma have also been shown to aid in cryopreservation and post-thaw viability. Although functions and roles of seminal plasma have been studied at length, the actual biochemical composition of sperm cells is poorly understood. In the present study, intra-species comparison of lipids, carbohydrates, and proteins present in sperm were examined in the bovine. Experiments were done in an effort to determine how cryopreservation may be affected by altering the level of cryoprotectant present in a sample based on baseline seminal composition of that particular specie. A wide variety of bovine breeds were used, representing both British and continental beef breeds as well as dairy. A portion of each sample was centrifuged to remove the cellular portion of the sample and both aliquots weighed to determine the cellular component’s contribution to weight of the sample. Results demonstrated a significant weight gain in equal volumes of each animal’s seminal plasma once the cellular component was removed (P < 0.001). Samples were then subjected to protein, carbohydrate, and lipid analysis. Each sample was run under spectrophotometric assay and blood chemistry assay cartridges to compare and contrast testing techniques. Differences in testing technique were detected in triglycerides and total protein levels (P = 0.023 and P = 0.018). Further, breed differences were also shown to be significant (α = 0.05) in triglycerides, glucose, total protein, and fructose (P = 0.001, P = 0.001, P = 0.001, and P = 0.009). However, no significant differences were seen between breeds for cholesterol (P = 0.228), as well as no differences detected in testing technique in cholesterol and glucose (P = 0.648 and P = 0.884 respectively). A non-linear correlation was observed between volume weights and total protein and triglyceride levels (P = 0.047 and P = 0.003). Together, these data suggest a difference in seminal component of various breeds and potentially on an individual basis as well. Further study is needed in order to examine how differences in seminal plasma chemistry effect cryopreservation and if it is possible to adjust cryoprotectant level in an effort to improve post-thaw viability of cryopreserved bovine semen.Item Design, synthesis, and calorimetric studies on protein-ligand interactions : apolar surface area, conformational constraints, and cation-[pi] interactions(2013-05) Myslinski, James Michael; Martin, Stephen F.Because bimolecular interactions in water are poorly understood, three tactics commonly used to improve binding affinity in ligand design were investigated: (1) increasing apolar surface area, (2) introducing a conformational constraint, and (3) targeting a cation-[pi] interaction. Thermodynamic parameters of binding ligands to the Grb2 SH2 domain were determined by isothermal titration calorimetry (ITC), and structural data was obtained by X-ray crystallography. The apolar surface area of the pTyr+1 residue in Ac-pTyr-Acnc-Asn-NH₂ was varied by incrementally increasing the size of the cyclic Acnc residue from a 3-membered to a 7-membered ring. Increasing apolar surface area resulted in an increase in Ka due to a more favorable [delta]H⁰ that was dominated a less favorable [delta]S⁰. Structural analyses showed that all ligands bound in a similar mode, so differences in binding thermodynamics were attributed to the pTyr+1 residue. The thermodynamics of binding tripeptides wherein pTyr+1 was an n-alkyl group were studied. Ka increased when Ala was mutated to Abu, but additional methylene groups had no effect on Ka due to strong entropy-enthalpy compensation. While [delta]H⁰ was weakly correlated with buried surface area, there was no change in [delta]H⁰ between one methylene and two methylene groups, presumably because an enthalpic penalty is associated with a gauche interaction between C-[beta] and C-[gamma] of the Xaa side chain that was noted in the crystal structure. An olefin was installed in an attempt to alleviate the energetic penalty incurred from the gauche interaction, but the introduction of the constraint resulted in equipotent ligands. A putative cation-[pi] interaction between Arg67 and various aromatic groups was probed by varying the [pi]-donating capability of groups attached to a tripeptide scaffold. Although crystal structures demonstrated that three of the aryl groups were close enough to Arg67 to form a cation-[pi] interaction, only a modest increase in Ka was observed relative to analogues having only an N-acetyl group. Furthermore, a simple cyclohexyl group in place of aryl groups resulted in ligands that were equipotent with indolyl- and phenyl- derived analogues, so any cation-[pi] interaction is not significant.Item DNA target site recognition and toward gene targeting in mammalian cells by the Ll.LtrB group II intron RNP(2013-05) Hanson, Joseph Haskell; Lambowitz, AlanMobile group II introns insert site-specifically into DNA target sites through a mechanism ("retrohoming") that involves reverse splicing of the intron RNA into the DNA and its subsequent reverse transcription by an intron-encoded protein (IEP) that is associated with the RNA in a ribonucleoprotein (RNP) complex. Characterization of this RNP complex and its retrohoming activities have enabled the development of programmable mobile group II intron gene targeting vectors routinely used in prokaryotic organisms. Building upon recent research by our lab to develop gene targeting in Xenopus laevis and Drosophila melanogaster using the group II intron Ll.LtrB from Lactococcus lactis, I describe work to extend this system to mammalian cells. I demonstrate that group II intron RNPs can be delivered to mammalian cells efficiently and produced in vivo via a CMV/T7 hybrid expression system. Using a robust single-strand annealing assay to detect homologous recombination induced by double-strand breaks (DSBs), I found that group II intron-mediated DSBs are efficiently repaired by mammalian cells. Despite varied approaches, I failed to detect endogenous group II intron-mediated gene targeting in human and mouse cells in culture. Gene expression microarray analysis and in vivo imaging of RNP molecules indicated that group II intron RNPs are sequestered away from the genome and induce host innate immune responses. I also investigated how the C-terminal DNA-binding domain of the Ll.LtrB IEP contributes to DNA target site recognition. Building upon previous mass spectrophotometric analysis of site-specific UV-crosslinking, I used genetic and biochemical analyses to identify potential protein contacts for key target site residues T-23 and T+5. Genetic selection of mutants in a region contacting T+5 led to identification of LtrA variants with increased retrohoming efficiency. My results provide evidence that the DNA-binding domain of a group II intron reverse transcriptase functions in DNA target site recognition and suggest new methods for changing its DNA target specificity and targeting efficiency.Item Evaluation of an oxygen delivering mobile perfusion device for organ transport(Texas Tech University, 2003-12) Cotter, Pete ZFew changes have been made to the clinical practice of donor heart preservation over the last 20 years. Using this protocol, viable storage of ex-vivo hearts is limited to no longer than 4 to 6 hours. In the present study, an oxygen delivering mobile perfusion device (ODMPD) was tested to determine if it could extend the ex-vivo heart viability using the porcine model. The first set of experiments was conducted to get baseline data. Three cadaver hearts were collected and stored for varying lengths of time to simulate different storage times using the conventional simple immersion method before being placed in the ODMPD for a minimum of 18 hours while monitoring pH, dissolved oxygen levels, and solution temperature. The second set of experiments was designed to test the ODMPD under real clinical conditions using a porcine model. A series of 13 experiments was conducted using the porcine model to simulate the conditions that the ODMPD would be expected to perform under in a human surgical setting. These experiments required the heart to be in the ODMPD from 8 to 24 hours, and in 2 cases transported in an airplane or in an automobile to simulate how they would be transported for clinical use. After all of the experiments, tissue samples were removed from the myometrium of the left ventricles and either fixed for electron microscopy or snap frozen for biochemical analysis. The biochemical analysis included testing for ATP, GSH, free iron, TBARs, and lipid radicals. After all of the tests were performed, the samples were separated by functional outcome of the transplantation and analyzed by group to determine correlations between specific biochemical levels and functional outcomes. As functionality decreased there was a positive relationship between increase in free iron (r = 9961), lipid radicals (r^2 =9995), and GSH (r^2 =8842). As functionality decreases, ATP levels decrease (r^2=.7961). These observations were supported by microscopic observations. Although this is preliminary data, it suggests that the ODMPD can both extend the viable life of the organ and could potentially facilitate the harvest of organs that were once thought to be marginally damaged by slowing the degradation processes that would render the organ useless.Item Relative contribution of insect and plant sugars to cotton fiber stickiness(Texas Tech University, 2003-05) Speck, Carl ThompsonNot availableItem Solid-supported phospholipid bilayers: separation matrix for proteomics applications(2009-05-15) Diaz Vazquez, Arnaldo JoelThis dissertation focuses on the development of biological platforms on which the function and characterization of transmembrane proteins can be performed simultaneously utilizing a biomembrane mimic consisting of a solid supported phospholipid bilayer (SLB). The study centered on the platform development, biophysical measurements of transmembrane proteins and membrane species chromatography. Membrane proteins play an essential role in various cellular and physiological processes. Their normal functions are essential to our health, and many impaired proteins have been related to serious diseases. Gaining a better understanding of membrane proteins is an essential step towards the development of more specific and competent drugs. This research study is divided into two main parts. The first part centered on the creation of a new platform for allowing transmembrane proteins to freely move inside supported lipid bilayers with the same mobility that can be found in vesicle systems. SLBs have been extensively used as model systems to study cell membrane processes because they maintain the same two-dimensional fluidity of lipids within the membrane found in live cells. However, one of the most significant limitations of this platform is its inability to incorporate mobile transmembrane species. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the protein, but also passivate the underlying substrate to reduce non-physiological protein-substrate interactions. High diffusion constants and high mobile fractions were obtained for a transmembrane protein reconstituted within this double cushion system. The second area of this study focuses on the creation of a new method to rapidly separate membrane components using electrophoresis in SLBs. This work showed that even subtly different chemical isomers can be well-separated by a simple electrophoretic technique when cholesterol is present in the separation matrix. As a first step towards the purification of proteins, this work showed that streptavidin proteins doubly bound to a bilayer by a biotinylated lipid can be separated from streptavidin proteins which are singly bounded.Item Structure, function, and inhibition of enoyl reductases(2009-05-15) Kuo, Mack RyanMalaria and tuberculosis constitute two of the world?s deadliest infectious diseases. Together, they afflict over one third of the world?s population. Once thought of as one of a group of nearly vanquished diseases only 50 years ago, malaria and tuberculosis have experienced renewed prominence due to issues such as multi-drug resistance and a lack of responsiveness by the global community. Fatty acid biosynthesis has been shown to be an essential pathway to the causative organisms of malaria and tuberculosis. One integral component of the fatty acid biosynthesis pathway, enoyl acyl-carrier-protein (ACP) reductase, has repeatedly been validated as an appropriate drug target in other organisms. The 2.4 ? crystal structure of the enoyl-ACP reductase from the human parasite Plasmodium falciparum (PfENR) reveals a nucleotide-binding Rossmann fold, as well as the identity of several active site residues important for catalysis. The 2.43 ? crystal structure of PfENR bound with triclosan, a widely utilized anti-bacterial compound, provides new information concerning key elements of inhibitor binding. Applying knowledge attained from these initial crystal structures, several triclosan derivatives were synthesized, and subsequently PfENR:inhibitor co-crystal structures were determined to extend our knowledge of protein:inhibitor interactions within the active site. Additionally, the crystal structures of the enoyl-ACP reductase from the mouse parasite Plasmodium berghei (PbENR), in apo-form and in complex with triclosan, were refined to 2.9 ? and 2.5 ? resolution, respectively. These structures confirm the structural and active site conservation between the human and mouse parasite enoyl-ACP reductases, suggesting that utilizing a murine model for in vivo testing of promising inhibitors is viable. The 2.6 ? crystal structure of the enoyl-ACP reductase from Mycobacterium tuberculosis (InhA) in complex with triclosan reveals a novel configuration of triclosan binding, where two molecules of triclosan are accommodated within the InhA active site. Finally, high-throughput screening approaches using enoyl acyl-carrier-protein reductases as the targets were utilized to identify new lead compounds for future generations of drugs. The 2.7 ? crystal structure of InhA bound with Genz-10850 confirms the value of this technique.Item Structure-function studies with the cAMP receptor protein of Escherichia coli(Texas Tech University, 2003-05) Tutar, YusufCyclic AMP Receptor protein (CRP) regulates the transcription of more than 100 genes. In the absence of cAMP, CRP is inactive. Cyclic AMP binding induces a structure change in CRP that promotes its interaction with RNA polymerase and DNA. CRP is a dimer of identical subunits; each consisting of 209 amino acids. A CRP subunit is composed of two domains. The larger N-terminal domain binds the allosteric effector, cAMP. This domain consists of eight j8-sheets that provide a hydrophobic pocket for cAMP binding. One cAMP is bound to each subunit contacting amino acid residues from both subunits. The C-terminal domain contains a helix-tum-helix motif that binds specific DNA sequences. The structure of CRP in the absence of cAMP is unknown, therefore the details of the allosteric mechanism mediated by cAMP remain obscure. The allosteric conformational change in CRP upon binding cAMP can be understood by comparing CRP and the CRP-cAMP complex by similar biophysical characterization. Several groups have used Raman spectroscopy, and circular dichroism techniques to compare these two different states of CRP. These methods used high concentrations of salt to improve CRP solubility. Our laboratory has used Fourier Transform Infrared Spectroscopy (FTIR) along with STIR cards to overcome the problems of protein solubility and high salt concentration. Analysis of the Amide I region indicated a secondary structure distribution of 35% a-helix, 31% jS-sheet, 21% turn, and 13% unordered for both states of WT and its E72D, E72Q, and R82Q mutants. This result is consistent with X-ray analysis of CRP-CAMP2 (37% a-helix, 36% ^-sheet). Fluorimetric binding studies showed that cAMP binding exhibits negative cooperativity in cAMP binding to the second subunit and amino acid substitution at positions 72 and 82 reduced binding affinities for cAMP by factors of 2 to 25 fold. DNA binding studies indicated that the equilibrium constants of the mutant CRP: cAMP complexes measured for lac? were reduced compared to that of WT CRP: cAMP complex. In addition, the mutant complexes failed to footprint in the presence of RNA polymerase. The level of j8-galactosidase expression in the mutants varied depending on this negative allostery. Since, under the conditions utilizied in this study, cAMP makes no contact with the DNA-binding domains, it cannot induce a conformational change in them by direct interaction. This suggests that cAMP induces a change in the relative orientation of the two subunits because it binds close to the subunit interaction area. This change could be relayed to the DNA binding domain and could change the relative position and orientation of the recognition helices and the activity. Thus these results can explain the allosteric transition mediated by the binding of cyclic AMP that converts CRP from a protein having low DNA activity to one that exhibits high, sequence-specific, affinity for DNAItem The coagulation of ovalbumin by shaking I. sulfhydryl titrations(Texas Tech University, 1964-05) Burton, James Arnold.Item Titanium-mediated Carbometallation of Homoallylic Alcohols(2012-07-20) Peng, Bo; Ready, Joseph M.Addition of zinc dichloride as an additive allows for the titanium-mediated carbometalation of homoallylic alcohols with Grignard reagents. The zinc dichloride additive successfully inhibits the B-hydride elimination of the titanium intermediate. The unsaturated products are obtained in up to 90 percent yield and up to >20 to 1 ratio of the carbometallation to oxidative arylation products. Subsequent electrophilic trapping is possible with elemental halides to yield the respective halohydrin products or oxidation to yield the diol products. The reaction is tolerant of both alkyl and aryl substituents on the homoallylic alcohol. This reaction allows ready access to unsaturated secondary alcohols or further functionalized products from an initial homoallylic alcohol.