Physical Sciences and Mathematics Commons^™

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 …

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 …

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 …

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 …

Isolation, Purification, Scanning Electron Microscopy And Bacterial Dna Uptake Of Plant Protoplasts, Bronwyn G. Hughes

Theses and Dissertations

Protoplasts were isolated from tobacco and barley leaves in sucrose or mannitol using commercially available cellulases and macerozymes. Barley growth and protoplast isolation and purification conditions were optimized so that protoplasts were obtained in high yields free of unwanted debris and organelles. A technique for processing barley and tobacco protoplasts for examination by scanning electron microscopy was developed in which protoplasts seem to have maintained their structural integrity. Barley and tobacco protoplasts took up 3H-B. subtilis DNA, 125I-B. subtilis DNA or 125I-M. luteus DNA as a linear function of time (0-6 hr) and DNA concentration (0-200 μg/ml). Up to 16 …

Studies Of N⁵, N¹⁰-Methylene Tetrahydrofolate Reductase From Porcine Kidney And Mouse L1210-Induced Tumor Tissues, Purification And Interaction With Antifolates, David W. Jayme

Theses and Dissertations

Methylene tetrahydrofolate reductase has been purified 1000-fold from porcine kidney and 400-fold from mouse L1210-induced tumor tissue by classical methods. The enzyme preparations have been demonstrated to be essentially free of contaminating methionine synthetase and serine transhydroxymethylase activity. Studies of the kinetic properties of the kidney and tumor enzymes, with respect to the reverse reaction using N5-methyl tetrahydrofolate as the variable substrate, have indicated Km values of 2.0 and 2.4 x 10-4 M, respectively. Inhibition of this key branch point enzyme in folate metabolism by a number of antimetabolites indicates that several of these antifolate compounds exhibit enzyme inhibition superior …

I. An Isothermal Titration Microcalorimeter. Ii. Importance Of Micronutrients In Nutrition. Iii. Zinc Bibliography, John Willard Gardner

Theses and Dissertations

I. An isothermal titration microcalorimeter having a colume of 4 ml and capable of temerature control to ±2 x 10-5°C is described. Major components include a constant temperature water bath controlled to ±3 x 10-4°C, a platinum reation vessel, and an isothermal control circuit consisting of constant Peltier thermoelectric cooling and variable Joule heating controlled by a thermistor in an AC Wheatstone bridge circuit. The calorimeter was tested by measuring the heat of ionization of water and was found to produce data accurate to ± 0.1% where small samples are used such as in the investigation of many biological systems.

Transforming Ability Of Bacillus Subtilis Dna Taken Up By Barley Embryos, Christine Tolman Ence

Theses and Dissertations

Purified Bacillus subtilis DNA taken up by one-day-old barley embryos and permanently fixed within the barley cell is shown to have maintained its biological activity in B. subtilis transformation. Cesuim chloride density gradient centrifugation of DNA isolated from the roots of barley embryos after the uptake and incorporation of radioactive, bromouracil-labelled B. subtilis DNA indicates the presence of the bacterial DNA in several discreet bands of well-defined density, whose density is changed by sonication. These preliminary findings concerning the fate of bacterial DNA taken up by barley roots are discussed in terms of the recombination model of Ledoux and Huart …

Enzymatic Synthesis Of Oligoribonucleotides Of Defined Base Sequence, Bronwyn Geraldine Hughes

Theses and Dissertations

A possible method for the synthesis of ordered oligoribonucleotides involves primer-dependent polynucleotide phosphorylase (PNPase) synthesis using the RNase A or T1 resistant N-cyclohexyl-N'-β(4-methylmorpholinium)ethylcarbodiimide chloride (CMC-Cl) derivatives of uridine or guanosine containing primers. Thus, CMC-UpA, CMC-GpA, CMC-UpCpC, CMC-GpApC, and ApApApApU-CMC were prepared and studied as primers for PNPase in 15 min 14C-ADP polymerization reactions and also in 6-10 hr synthesis reactions. The CMC-primers were not suitable primers for PNPase catalyzed synthesis reactions for either time. The PNPases used in such studies were contaminated with nuclease that showed the following order of susceptibility: UpA > CpC, GpA > ApU, ApA. Even a highly purified …

A Study Of Folic Acid Dependent Reactions In Bovine Brain, Dillard Spencer Broderick

Theses and Dissertations

The enzymatic interconversion of serine to glycine and N5, N10-methylene tetrahydrofolate to N5 -methyl tetrahydrofolate have been studied in a cell-free system derived from bovine brain. A partial purification of serine transhydroxymethylase and N5, N10 -methylene tetrahydrofolate reductase has been achieved by ammonium sulfate fractionation, protamine sulfate fraction, and by various column chromatography procedures. The two enzymes studied catalyze sequentially the reductive transfer of the hydroxymethyl group of serine to the N5 position of tetrahydrofolate forming N5 -methyl tetrahydrofolate. The products of these reactions were assayed by the incorporation of C14 from a one-carbon precursor i.e., L-beta-(C14)-serine, or N5 -methyl …

The Role Of Thiamine Pyrophosphate In Regulating The Level Of Gamma-Aminobutyric Acid In The Central Nervous System, Bryant L. Adams

Theses and Dissertations

The level of gamma-aminobutyric acid in the brains of white rats suffering from thiamine deficiency symptoms resulting from pyrithiamine or oxythiamine antagonism of thiamine or from thiamine deprivation was measured. The level of this amino acid was 17.5% lower in the brains of pyrithiamine-treated rats and 20.0% lower in the brains of thiamine-deprived rats than in the control rats. Daily injections of ten times the required amounts of vitamin B6 in rats treated with pyrithiamine or oxythiamine or by thiamine deprivation had no effect on the level of brain gamma-aminobutyric acid or on the psiological behavior of these rats. The …

Studies Of Rat Brain Thiamine Pyrophosphokinase, Lavell Rolfson Johnson

Theses and Dissertations

The enzyme ATP:thiamine pyrophosphotransferase (thiamine pyrophosphokinaseJ was purified from acetone powders which were prepared from rat brains up to the chromatography on alumina C_γ step by a modified procedure that Mano (Y. Mano, J. Biochem., 42, 283 (1960)] developed for the purification of the rat liver thiamine pyrophosphokinase. The enzyme was purified 12 fold over that in the original extract of the acetone powder. A spectrophotometric assay for the brain thiamine pyrophosphokinase was developed utilizing brewer's yeast apotransketolase. The transketolase assay was able to detect less than 10^-11 moles of thiamine diphosphate, which was synthesized by the kinase, in the …

The Vitamin B1-Sparing Action Of Sorbitol, Gisela Eleanor Bethsold

Theses and Dissertations

The present study was initiated to determine the effect sorbitol has upon the enzymatic functions of thiamin; specifically, what effect sorbitol bas upon the alpha-ketoglutarate and pyruvate dehydrogenases activities of rat liver mitochondria. In addition, the effect of varying the dietary sorbitol concentration on the development of an athiaminosis was studied, particularly the effect on growth rate.

Studies On The Biological Oxidation Of Ferrous Iron And Chalcopyrite, Loral Alvin Decker

Theses and Dissertations

The purpose of this investigation was to determine the effectiveness of the leaching streams from waste copper ore dumps as a nutrient solution for subsequent biological oxidation and solubilization of sulfide minerals. The copper in the leaching streams is precipitated by displacement with iron. As a result of this procedure the solution is high in ferrous iron concentration and has a pH of near 3.7. The bacteria in the leaching streams are capable of oxidizing ferrous iron to the ferric state and sulfide minerals to sulfates. The effects of temperatures, pH, urea, and aeration rate on the oxidation of the …

A Study Of Three Photochemical Activities Of Chloroplasts, Jimmy W. Hinkson

Theses and Dissertations

Chloroplasts of sugar beet leaves were subjected to treatment by heating, by incubation at pH 8.5 and pH 5.5, by incubation in ion low water, by sonic oscillation, by digestion with enzymes (lysozyme, pancreatin, and lipase), by extraction with digitonin, and by chemical compounds including 8-hydroxy-quinoline, sodium azide, zinc sulfate, phenyl mercuric acetate, potassium cyanide, 1-allylthiourea, 2,4-dinitrophenol, thymol, and p-chloromercuribenzoate. A comparative study was made of the effects of these treatments upon three photochemical reactions (the photoreduction of 2,6- dichlorophenol indophenol, the photoreduction of indigo carmine in the presence of a couple composed of 2,6-dichlorophenol indophenol and ascorbic acid, and …

Chemosynthetic Microorganisms In Leaching Sulfide Minerals, A. Keith Jameson

Theses and Dissertations

This investigation showed that a single bacterium was capable of sulfide oxidation in exposed ore bodies. This single bacterium was isolated and characterized. It exhibited different characteristics than any previously known organism. It was found to be a chemosynthetic autotroph. An optimum nutrient concentration was determined for pyrite oxidation. The nutrient concentration was determined for pyrite oxidation. The nutrient contains only a source of phosphate and nitrogen. Urea was found to be a better source of nitrogen than ammonium sulfate which had previously been used. The effect of various organic compounds on the oxidation of pyrite was observed. Glucose and …

Oxidation Of Molybdenite With The Aid Of Microorganisms, Ralph Anderson

Theses and Dissertations

This work represents a study on the biological oxidation of molybdenite, MoS2. The principal objective of this study was to investigate the possibility of the biological oxidation of molybdenite, to determine the physical and chemical conditions under which the oxidative process occurs, and to develop a strain of bacteria or alter the activity of the microorganisms by acclimatization to increase their activity on sulfide minerals, in particular molybdenite. Minerals used in this study were pyrite (FeS2), chalcopyrite (CuFeS2), molybdenite concentrate, and molybdenite ore. The microorganisms used in this investigation were autotrophic bacteria obtained from the leaching streams of Bingham Canyon, …

Photo-Oxidations Sensitized By Physiologically Important Substances, Ernel D. Ihnen

Theses and Dissertations

A study of the photo-oxidation of the 2,6-dichlorophenol indophenol-ascorbic couple was undertaken with a view to investigating the postulated mechanism of oxidation by a component of the oxygen evolving sequence operative in photosynthesis. A spectrophotometric method for the measurement of the progress of photo-oxidation was developed. Temperature and pH dependence were determined and the effect of various common inhibitors and physiological substances tested. None of the common Hill reaction inhibitors were effective in the ascorbic acid photo-oxidation in the system used. KCN and catalase as well as a few other high molecular weight proteins were able to inhibit dark auto-oxidation …

The Chromatographic Purification Of Human Red Blood Cell Acetylcholinesterase, Richard P. Dunkley

Theses and Dissertations

A process of column chromatography of human erythrocyte acetylcholinesterase, using a cationic exchange resin, was investigated as a possible method for the primary partial purification of the enzyme on a large scale. The investigation was based, first; on a detailed study of the enzymatic stability to variation in hydrogen-ion and salt concentrations for periods of time extending to forty eight hours and, second; on an investigation of the degree of adsorption by the resin of the enzyme at various pH values within the pH range of enzyme stability. A method for the analysis of the enzyme activity of large numbers …

Biological Oxidation Of Copper Sulfide Minerals, Delmar Boyd Davis

Theses and Dissertations

This work represents a study of the biological oxidation of copper sulfide minerals. The principal objective of the study was to determine the extent of bacterial oxidation on copper containing sulfide minerals. It was also desired to determine if the bacteria could oxidize copper sulfides in the absence of iron. A better understanding of the nitrogen requirements of the organisms was desired. Minerals used in this study were Bingham Canyon Float Concentrate, chalcopyrite, covellite, calcocite, bornite, tetrahedrite, and reagent grade copper sulfide. The bacteria used were obtained from the mine waters of Bingham Canyon, Utah. To study the problem the …