Biochemistry, Biophysics, and Structural Biology Commons^™

An Acyl Radical Cascade Model For The Total Synthesis Of Lyconadin A, Seth W. Grant

Theses and Dissertations

Lyconadin A (1) is a structurally unique Lycopodium alkaloid with antitumor properties, isolated from the club moss Lycopodium complanatum. We are developing a synthetic route to 1 based on a novel 7-exo-trig/6-exo-trig acyl radical cascade cyclization. The synthesis of model acyl radical cascade precursor 23 will be presented. Key features of this synthesis include the suppression of competing elimination during the alkylation of a hindered phenethyl bromide and the use of a lactone as a precursor to a compound bearing two differentially protected primary alcohols. An account of our studies on the model acyl radical cascade cyclization (23 to 24 …

Effect Of Melatonin And Dopamine In Site Specific Phosphorylation Of Phosducin In Intact Retina, Arinzechukwu Okere Nkemdirim

Theses and Dissertations

Phosducin (Pdc) is a 28 kDa binding partner for the G protein beta gamma subunit dimer (G-beta-gamma) found abundantly in the photoreceptor cells of the retina and pineal gland. In the retina, light-dependent changes in cAMP and Ca2+ control the phosphorylation of Pdc at serine 73 and 54, respectively, which in turn controls the binding of Pdc to G protein beta gamma subunit dimer . G protein beta gamma subunit dimer binding has been proposed to facilitate light-driven transport of G protein beta gamma subunit dimer from the site of phototransduction in the outer segment of the photoreceptor cell to …

Angiotensin Ii-Mediated Regulation Of The Human Angiotensin Ii Type 1 Receptor Gene, Xylophone Vijai Aasee Victor

Theses and Dissertations

The physiological responses of angiotensin II (Ang II) are mediated across the cell membrane through the angiotensin II type 1 receptor (AT1R), a heptahelical membrane protein coupled to trimeric G-proteins on the cytosolic side. AT1R on binding its ligand, Ang II, leads to downregulation of cell-surface receptor and also its mRNA. We have investigated whether the 3'- and 5'-untranslated regions of the human AT1R mRNA mediate the degradation of hAT1R mRNA by post-transcriptional mechanisms in human adrenocortical carcinoma cell line (H295R cells). Protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA), showed that the downregulation of hAT1R mRNA is mediated by the …

Development Of A Model For The Kinetics And Mechanism Of Nitrogenase, Phillip E. Wilson

Theses and Dissertations

Nitrogenase has a central role in the global nitrogen cycle as the enzyme that catalyzes the reduction of atmospheric N2 to NH3. Fixed nitrogen is generally limiting in the environment and in agriculture, so nitrogenase has received much attention as an alternative to nitrogen fertilizers. Characterizing the mechanism of nitrogenase is the goal of this work. The molybdenum nitrogenase enzyme system is comprised of the MoFe protein and the Fe protein. Interactions between these proteins and nucleotides are crucial to catalysis. An important approach to characterize these interactions is to correlate the kinetics of nitrogenase catalysis to a mechanism based …

Alterations In Uterine And Placental Sodium Pump Abundance May Contribute To The Onset Of Mouse Labor, Carlos Jacob Vance

Theses and Dissertations

Objective: Reductions in sodium pump (SP) abundance can give rise to increases in contractile force in uterine and vascular smooth muscle as well as an increased secretion in secretory cells, including potentially those of the placenta. To determine whether the mouse might serve as a model for human pregnancy in terms of the SP and to determine whether changes in SP abundance anticipate or follow labor, we studied pregnant mice over the final trimester of their pregnancy.

Study Design: C57Bl6 dams (n=46) were bred and studied during their pregnancy. Animals (n=4) were sacrificed at specific gestational time points. Other mice …

Ion Transmission In The First Vacuum Stage Of An Inductively Coupled Plasma Mass Spectrometer, Jeffrey Harris Macedone

Theses and Dissertations

The inductively coupled plasma mass spectrometer (ICP-MS) is the instrument of choice for trace and ultra-trace elemental analysis. However, the ICP-MS suffers from matrix effects. Matrix effects occur when instrument response varies as the composition of the sample matrix is changed. Matrix effects, or non-spectroscopic interferences, limit the accuracy of routine analysis. Identification of the sources of matrix effects provide a basis for reducing or eliminating them. As inaccuracies in the ICP-MS are more severe than those in the ICP atomic emission spectrometer, the problem may be due, at least in part, to the vacuum interface used to couple the …

Identification Of The Pla2 Responsible For Prostanoid Synthesis In Response To Inflammatory Cytokines, Chaminda Fernando

Theses and Dissertations

Preliminary studies from our laboratory showed that cPLA2α may be responsible for approximately 50-60% of the PGE2 production in response to inflammatory cytokines. Thus, we hypothesized that a closely-related PLA2 is responsible for 40-50% of the PGE2 produced in response to inflammatory cytokines. To this end, we utilized RNAi technology, extensively optimized, to down regulate the expression of closely-related isoforms of phospholipase A2 in A549 cells and used an enzyme linked immuno sorbent assay (ELISA) to quantitate the PGE2 produced. These studies found that cytosolic phospholipase A2α (cPLA2α) regulated 97.7% of the prostaglandin E2 (PGE2) produced in response to inflammatory …

Vimentin Overexpression Contributes To The Biological Properties Of Metastatic Head And Neck Cancer Cells, Rachel J. Paccione

Theses and Dissertations

Epithelial to mesenchymal transition occurs in the later stages of epithelial tumor progression, with cells expressing mesenchymal markers. Of these, the intermediate filament protein vimentin is frequently upregulated in metastatic carcinomas. Previously, microarray studies showed that the gene encoding vimentin is highly upregulated in metastatic HN12 cells compared to a related primary tumor cell line. In this study, we confirmed this difference using real-time quantitative PCR, western blot analysis, and immunostaining. Furthermore, EGF and TGF-β, growth factors that induce migration and invasion of HN12 cells, produced synergistic increases in vimentin expression. To assess the contribution of vimentin to the biological …

Modulation Of Gene Expression By Tumor-Derived Mutant P53. Role Of Transactivation In Gain-Of-Function., Mariano J. Scian

Theses and Dissertations

It was hypothesized that the C-terminal sequences for mutant p53 would be required for oligomerization. and oligomerization may be critical for gain-of-function. An N-terminal deletion mutant of p53 that deletes amino acids 1-293 was used as a tool to perform hetero-oligomerization studies. This mutant retains the entire oligomerization domain but dispenses off the transactivation domain and a large portion of the sequence- specific DNA-binding domain. Co-transfection experiments show that p53 del. 1-293 forms hetero-oligomeric complexes with p53-D281G. Also. co-expression of p53 del. 1- 293 with p53-D281G inhibited p53-D28lG-mediated transactivation of the EGFR and MDRl promoters suggesting that hetero-oligomerization inactivates transcriptional …

The Influence Of Lipid Composition On The Binding Of Ldl To Chondroitin 6-Sulphate, Wilma Espiritu

Theses and Dissertations

The interaction between low-density lipoprotein (LDL) and glycosaminoglycans is a key factor in atherosclerosis. The present study examines the characteristics of LDL and its binding properties with the main glycosaminoglycan of the vascular wall, chondroitin 6- sulphate (C6S). The compositional characteristics that were studied for each LDL sample were phase transition temperature, phospholipid content, free cholesterol content, cholesteryl ester content, triglyceride content, and size. Correlations of these characteristics with LDL-C6S binding were analyzed using a turbidity assay. Our results showed that there is no correlation between LDL-C6S binding and phase transition temperature, triglyceride content, or size. Strong correlations were present …

Identification Of The Rna Cis-Elements That Interact With Srp30a To Regulate The Alternative Splicing Of Caspase 9 Pre-Mrna, Prabhat Mukerjee

Theses and Dissertations

Studies have shown that the alternative splicing of caspase 9 and the phospho-status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and de novo ceramide via the action of protein phosphatase-1 (PP1). Two RNA splice variants are derived from the caspase 9 gene, pro-apoptotic caspase 9a and anti-apoptotic caspase 9b, via alternative splicing by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. In this study, the link between SR proteins and the alternative splicing of caspase 9 was established. Sequence analysis of the exon 3, 4, 5, and 6 …

Cytokine And Chemokine Profiles In A Rat Model Of Hemorrhagic Shock After Immuno-Modulation By Androstenetriol, Kristin E. Paccione

Theses and Dissertations

Further understanding of the cellular and molecular mechanisms involved in traumatic injury, and how they are modulated during drug interaction, can facilitate novel treatment strategies for future trauma patients. We hypothesize that the pharmacological agent, Androstenetriol (AET), up regulates host immune response by modulating the continued expression of mediators, including cytokines. In a double-blinded experiment, rats were hemorrhaged, driven by volume or pressure conditions, then resuscitated with fluids and packed red blood cells following a subcutaneous injection of either vehicle or drug. Blood was collected at various time points and cytokine levels were determined by analyses with both multiplex and …

Capillary Liquid Chromatography Using Micro Size Particles, Yanqiao Xiang

Theses and Dissertations

High speed and/or high efficiency separations can be realized using small particles (~ 1 µm) in liquid chromatography (LC). However, due to the large pressure drop caused by small particles, conventional LC pumping systems cannot satisfy the pressure requirements needed to drive the mobile phase through the column. Use of ultrahigh pressure, elevated temperature, or both can overcome these pressure limitations and allow the use of very small particles for high speed and/or high efficiency separations.

In this dissertation, the use of ultrahigh pressures with and without elevated temperatures in capillary LC is described. Very fast separations of various samples …

Velocity And Temperature Characterization Of The First Vacuum Stage Expansion In An Inductively Coupled Plasma - Mass Spectrometer, William Neil Radicic

Theses and Dissertations

The inductively coupled plasma - mass spectrometer (ICP-MS) is the analytical instrument of choice for trace element detection and quantification. Despite the popularity of ICP-MS, significant degradation in sensitivity and precision occurs as the result of matrix and instrument-induced effects. The sources of these effects are not well understood, characterized, or correlated to particular plasma operating condition settings or matrix compositions and involve both neutral and charged species. The purpose of this study is to characterize the behavior of metastable Ar (I) atom and Ca (II) ion through the measurement of Doppler velocities and fluorescence line width "temperatures."

For the …

N9 Alkylation And Glycosylation Of Purines; A Practical Synthesis Of 2-Chloro-2'-Deoxyadenosine, Minghong Zhong

Theses and Dissertations

(a) The Robins reagent [2-acetamido-6-O-(diphenylcarbamoyl)purine] was utilized for glycosylation under Lewis acid conditions. Regioselectivity of glycosylation depends on the glycosyl donor and its 2-O- or 2-N-protecting group. Regioselective N9 glycosylation of 2-acetamido-6-O-(diphenylcarbamoyl)purine with problematic glucosamine has been accomplished by protecting the amino function as a phthalimido group with consequent stabilization of the oxocarbenium cation, and lowering the activation energy by introduction of trichloroacetimidate at the anomeric carbon.

(b) 6-Heteroaryl functions [6-(1,2,4-triazol-4-yl) and 6-(imidazol-1-yl)] were introduced into purine derivatives for regioselective N9 alkylation. The regiospecificity of alkylation mainly results from steric effects due to the coplanar conformation of the two linked …

Fluorescence Detectors For Proteins And Toxic Heavy Metals, Uchenna Prince Paul

Theses and Dissertations

An inexpensive detector for proteins is described. The detection technique was based on two-photon excitation intrinsic protein fluorescence using a visible 532 nm diode-pumped nano laser as the excitation source. Proteins that exhibit intrinsic fluorescence must contain at least one tryptophan, tyrosine, or phenylalanine residue in their amino acid sequences. The detector was characterized and was found to have a detection limit of 4 micro-molar for tryptophan, 22 micro-molar for tyrosine and 500 micro-molar for phenylalanine. Bovine serum albumin, a serum protein with 3 tryptophan residues in its amino acid sequence was also used to characterize the detector. It was …

Analysis Of Clinically Important Compounds Using Electrophoretic Separation Techniques Coupled To Time-Of-Flight Mass Spectrometry, Zlatuse Durda Peterson

Theses and Dissertations

Capillary electrophoretic (CE) separations were successfully coupled to time-of-flight mass spectrometric (TOFMS) detection for the analysis of three families of biological compounds that act as mediators and/or indicators of disease, namely, catecholamines (dopamine, epinephrine, norepinephrine) and their O-methoxylated metabolites (3-methoxytyramine, norepinephrine, and normetanephrine), indolamines (serotonin, tryptophan, and 5-hydroxytryptophan), and angiotensin peptides. While electrophoretic separation techniques provided high separation efficiency, mass spectrometric detection afforded specificity unsurpassed by other types of detectors.

Both catecholamines and indolamines are present in body fluids at concentrations that make it possible for them to be determined by capillary zone electrophoresis coupled to TOFMS without employing any …

Cell Modeling, Matthew L. Campbell

Theses and Dissertations

The Air Force is currently developing new products that incorporate a variety of chemicals which may come in contact with product users. To define which chemicals are dangerous to the user, toxicity studies have been performed. However, analysis of toxicity ultimately requires models of the exposed cellular systems. This thesis provides an introduction of how to model and analyze small and large cellular systems. Understanding the underlying behavior of small models and their relation to large systems will lead to a better understanding of how the Air Force should construct intracellular models to assist in future toxicology studies. Developing analysis …

Comprehensive Isotachophoresis-Capillary Electrophoresis Coupled To Time-Of-Flight Mass Spectrometry, Christopher Ryan Bowerbank

Theses and Dissertations

Isotachophoresis (ITP) coupled to capillary zone electrophoresis (CE) in a comprehensive manner was used to separate mixture components in both insufficient and sufficient concentrations without heart-cutting or splitting. Examples of comprehensive ITP-CE involving multiple CE injections of preconcentrated ITP zones are demonstrated. In the comprehensive arrangement, all of the sample in the first dimension (ITP) is subjected to analysis in the second dimension (CE), without significant sample loss or decrease in sample detectability resulting from removal of a portion of the sample. This is especially important for analytes at low concentrations which may form a single mixed zone instead of …

Characterization Of Rat Pulmonary Carboxylesterase, Timothy J. Wallace

Theses and Dissertations

The 1839 bp cDNA for rat pulmonary carboxylesterase was cloned by reverse transcription polymerase chain reaction (RT PCR) from total rat lung RNA using specific primers derived from the 5' and 3' untranslated regions of rat hepatic cholesteryl ester hydrolase (CEH). The unique cDNA was sequenced and found to have 99% homology with hepatic CEH. This homology extends to the predicted amino acid sequences which show only six amino acid residue differences in the coding region: three conserved and three nonconserved changes. However, the catalytic activitites of the two proteins are dramatically different. While CEH hydrolyzes cholesterol oleate, the pulmonary …

Chemical Synthesis, Bacterial Expression, And Characterization Of Pro-Gnrh/Gap, A Precursor Protein Of Two Biologically Active Peptide Hormones, Louis E. Mcadory

Theses and Dissertations

Two biologically active peptides, gonadotropin releasing hormone (GnRH) and GnRH associated peptide (GAP) are both derived from a common prohormone precursor protein, pro-GnRH/GAP. Both peptides are cosecreted from hypothalamic neurosecretory granules and are involved in the regulation of mammalian reproduction. A calcium dependent, neutral pH serine protease discovered in this laboratory, GAP-releasing enzyme, is the most likely processing enzyme of pro-GnRH/GAP. GAP-releasing enzyme is immunologically related to PC1/3, a member of the prohormone convertase (PC) class of processing endoproteinases.

GAP-releasing enzyme recognizes the eight residue processing site within pro-GnRH/GAP, G⁶LRPGGKR¹³, and correctly cleaves the R^{13 …}

Molecular Cloning And Analysis Of The Promoter For Rat Hepatic Neutral Cholesterol Ester Hydrolase, Ramesh Natarajan

Theses and Dissertations

Neutral cholesterol ester hydrolase (CEH) is a key enzyme in regulating hepatic free cholesterol. Using the CEH specific cDNA sequence in the 5'-untranslated region as a primer, 1.3 kb of sequence upstream of the ATG initiation codon was amplified and cloned. Primer extension analysis with total RNA from rat primary hepatocytes identified a transcription initiation site, 60 bases upstream from the initiation codon. No typical TATA-box sequences were found upstream from the transcription start site. However, a consensus GC-box, which can bind the positive transcription factor SP1, was found 35 bases upstream from the transcription start site. In addition the …

Red Blood Cell Stability In Uremic Rats, Cecilia Mermel Maccallum

Theses and Dissertations

Determining the fragility of the red blood cell (RBC) is important for the diagnosis of and evaluation for treatment of several RBC diseases. In part RBC production is controlled through the hormone erythropoietin secreted by the kidney. In a previous study from this laboratory, it was found that RBC were more stable in uremic male rats compared to controls. In this experiment, uremia was induced in four groups of female rats through a two stage nephrectomy. The nephrectomy involved the removal of two-thirds of the left kidney, followed by the removal of the entire right kidney one week later. The …

Regulatory Analysis Of Vimentin Expression In Metastatic Versus Nonmetastatic Breast Cancer Cell Lines, Danielle N. Bird

Theses and Dissertations

The intermediate filament gene family, composed of six classes, shows both tissue and development-specific expression. Vimentin is a unique member of the intermediate filament protein family: although it is expressed in cells of mesenchymal origin, vimentin may also be expressed with other intermediate filament proteins during early stages of development. In some cases, as differentiation continues, vimentin is normally down-regulated whereas other intermediate filament proteins, like desmin, glial fibrillary acidic protein, or neurofilaments are turned on in muscle, glial cells, or neurons, respectively. In some metastatic cancers, including breast and prostate cancers, vimentin is aberrantly expressed, despite the embryonic origin …

Expression, Purification And Characterization Of Hepatitis C Virus Core Protein From E.Coli Using A Chemically Synthesized Gene (1), And Cloning, Expression, Purification And Characterization Of The Major Core Protein (P26) From Equine Infectious Anaemia Virus, Ashley James Birkett

Theses and Dissertations

The hepatitis c virus (HCV) core gene has been chemically synthesized and used to direct the expression of core protein in Escherichia coli (E.coli). When cloned downstream of the inducible T7 promotor the core gene directed the expression of a soluble protein with a molecular weight of 22 kDa. Under native conditions the protein behaved in a manner consistent with the formation of a multimeric structure, which may represent assembled core particles. Further, when examined by electron microscopy a heterogeneous mixture of nucleocapsid-like particles were visible. Core protein was specifically recognized by antibodies present in HCV infected serum, suggesting that …

Cloning, Expression, Purification And Characterization Of Domain B Of Wheat Germ Agglutinin, Ann C. Rice

Theses and Dissertations

Wheat germ agglutinin (WGA) belongs to a family of dimeric chitin binding lectins specific for N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-neuraminic acid (NeuNAc). The polypeptide chain consists of a tandem repeat of four conserved 4.3 kDa domains (A, B, C, D) stabilized by four disulfide bonds. Saccharide binding occurs in the dimer interface where domains of different subunits are in contact (A with D, B with C). An aromatic amino acid rich pocket on one domain (sufficient for saccharide binding) and a polar region on the contacting domain constitute a complete binding site. Saccharide binding affinities may differ among the four unique …

Biochemical Characterization Of The Axolemmal Mitogen For Cultured Schwann Cells, Mark A. Decoster

Theses and Dissertations

The molecule(s) involved at the axon plasma membrane (axolemma) which causes Schwann cells to proliferate has been investigated by three biochemical techniques: 1) Alkaline extraction of axolemma resulted in recovery of 95% of the mitogenic activity and 50% of the protein in the membrane-bound portion of the axolemma. 2) The axonal mitogen for Schwann cells may be associated with heparan sulfate proteoglycans at the axonal surface. Treatment of axolemma with heparitinase, which cleaves the glycosidic bonds of sulfated glycosaminoglycans, allowed mitogenic activity to be solubilized. 3) Treatment of axolemma with heparin, a highly sulfated glycosaminoglycan analogous to heparan sulfate, resulted …

Proliferation Of Schwann Cells Induced By Axolemmal And Myelin Membranes, Michael Paul Dinneen

Theses and Dissertations

Purified Schwann cells were cultured from neonatal rat sciatic nerve using a modification of the method of Brockes (Brockes, J.P. et al, Brain Res. 165(1979) 105-118). Schwann cells and contaminating fibroblasts were unambiguously identified using fluorescent antibodies to 2'3' cyclic nucleotide 3'-phosphodiesterase and the thy 1.1 antigen respectively. The Schwann cells were quiescent unless challenged with mitogens, They proliferated rapidly in response to the soluable mitogen, cholera toxin, or to membrane fractions from rat CNS or PNS, prepared by the method of DeVries (DeVries, G.H., J Neurochem, 40 (1983) 1709-1717). Mitogenic activity was present in both axolemmal and myelin enriched …

Nuclear Envelope Proteins: Chemical Characteristics And Molecular Interactions Of The Lamins, David Lee Cochran

Theses and Dissertations

The lamins are a group of proteins in a residual nuclear envelope fraction derived from the nuclear lamina. The absence of specific functional assays for the two predominant species, lamin A and lamin B, has complicated their biochemical characterization. Initial studies reported in this work involved the enzymic cleavage of isolated lamins A and B and another lamina protein with a molecular weight of 61,000-daltons. The results obtained with each of three enzymes indicated strong similarities between the three nuclear envelope proteins.

Further chemical characterization of the lamins has been achieved. Lamins A, B and C have been separated by …

Effects Of Pulsed Electric Fields On Mammalian Cell Membranes, Guy K. Smith

Theses and Dissertations

The effect of pulsed electric fields on cell membranes were studied to investigate the effects of an electric field, per se, on biological systems, without the densometric and other technical problems associated with other forms of nonionizing radiation. Exposure of mouse splenocytes to a high voltage pulse resulted in an increase in membrane permeability to K⁺ that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 μsec, 3.0 kV/cm pulse elicited a 50% loss of intracellular K⁺ indicating that the critical transmembrane potential (V_m) at breakdown was 1.26 volts …