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A Molecular Basis For Erythromycin Sensitivity And Resistance In Escherichia Coli, Harold S. Chittum

Electronic Theses and Dissertations

The effect of erythromycin on the 50S ribosomal subunit during cell growth has been extensively investigated. Sucrose density gradient analysis of ribosomes formed in the presence and absence of the drug revealed a 50S specific assembly defect is partially responsible for erythromycin's inhibitory effects on wild type cells. Examination of two erythromycin-resistant mutants of E. coli (N281 and N282) revealed that mutant N281 (L22 mutant) but not N282 (L4 mutant) was assembly defective in the presence of the drug, although only at much higher drug concentrations (300 ug/ml vs. 75 ug/ml for wild type cells). The altered genes from each …

A Characterization Of Extractable, Hydroxylated Fatty Acid Bearing Components In Legionella Pneumophila, Jonathan R. Lane

Electronic Theses and Dissertations

Extraction of the lipids of Legionella pneumophila yields phases unlike those produced from other Gram-negative bacteria. A viscous interface forms between the aqueous (wash) and organic phases. More than half of the hydroxylated fatty acids were found distributed between the aqueous phase and the interfacial material, fractions in which such constituents have not been reported in other Gram-negative species. It was further observed that after the material from the aqueous/interfacial phase was dissolved in methanol or chloroform/methanol (2:1 (V/V)), the addition of acetone would create a white, flocculent precipitate. Analyses showed that the supernatant contained fatty acids that were nonhydroxylated …

Human Lung Mast Cell Tryptase Isozymes: Separation And Examination Of Structural And Functional Differences, Susan S. Little

Electronic Theses and Dissertations

Tryptases are trypsin-like enzymes found in mast cell granules. Although in vivo substrates have not been positively identified, tryptases cleave a limited number of potential physiological substrates in vitro, including high molecular weight kininogen (HMWK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) apparently exists as a tetramer with an M$\sb{\rm r}$ of 135-144 kDa by gel filtration, whereas SDS-PAGE yielded two bands of M$\sb{\rm r}$ 29 Kda and 33 Kda. Tryptases are resistant to inhibition by most natural trypsin inhibitors and display some affinity for heparin. The existence of tryptase isozymes has been implied from …

Probing Protein-Protein Interactions Among Proteins Of A Nonaggregated Fatty Acid Synthetase From Euglena Gracilis Variety Bacillaris, Sande G. Williams

Electronic Theses and Dissertations

Enoyl-acyl carrier protein (ACP) reductase from chloroplast nonaggregated fatty acid synthetase (FAS) of Euglena gracilis variety bacillaris was purified to a single band on a denaturing polyacrylamide gel. The enzyme was partially characterized with respect to substrate specificity, reduced nucleotide requirement, and the effect of ACP and Ca$\sp{++}$ on enzyme activity. Antibodies against the purified protein were raised in hens and isolated from eggs. ACP was purified from Euglena in yields of about 1mg/100g (wet weight) of cells. Antibodies were raised against the purified protein. ACP antibodies inhibited the Euglena chloroplast FAS using Euglena or E. coli ACP as a …

Regulation Of The Yeast Transcriptional Activator Adr1, W James Cook

Doctoral Dissertations

Glucose repression of the ADH2 gene from Saccharomyces cerevisiae is mediated by the abundance and activity of the transcriptional activator ADR1. The focus of this dissertation is to characterize the mechanisms by which glucose controls the ability of ADR1 to activate ADH2 transcription.

Glucose results in a two-fold decrease in the steady state levels of ADR1 mRNA. This glucose-dependent reduction in steady state ADR1 mRNA was shown to be due to an increased rate of ADR1 mRNA degradation. The unusually long, 510 nucleotide 5$\sp\prime$ untranslated region of the ADR1 mRNA appeared to mediate this glucose-dependent ADR1 mRNA decay.

To better …

Equilibrium Electrophoresis: Results From The Second Prototype, David Barton Hayes

Doctoral Dissertations

Equilibrium electrophoresis is a new method that determines the apparent charge on macroions. The theory of equilibrium electrophoresis is summarized. The second generation device is described. Apparent charges, at various ionic strengths, measured by equilibrium electrophoresis are reported for pd(A)$\sb{20},$ pd(T)$\sb{20},$ pd(A)$\sb{20}$ $\cdot$ pd(T)$\sb{20},$ cytochrome c from horse heart, TA lysozyme mutant, TA(119/135) charge mutated lysozyme, and EDTA. The apparent charge of plasmid pBR322 from electrophoretic mobility and sedimentation velocity is reported. The results are compared to those expected according to the Record-Manning counterion condensation theory and the Debye-Huckel shielding theory. The apparent charges of the two ss DNA samples …

The Association Properties Of Retroviral Integration Protein, Jacqueline Lei Coleman

Doctoral Dissertations

Retroviral Integration Protein (IN) has been shown to be both necessary and sufficient for the integration of reverse transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-association is required for proper function, yet little is known about the thermodynamic properties of this association. Analytical ultracentrifugation was used to determine the stoichiometries and free energies ($\Delta$G) of IN self association under various physical conditions. The best fit model of association in all glycerol-free conditions studied was a reversible monomer-dimer-tetramer. The addition of glycerol at concentrations of 8% (vol/vol) or greater significantly enhances the monomer-dimer formation so …

Kinetics And Mechanism Of Iron Oxidation In Apoferritin, Shujun Sun

Doctoral Dissertations

The enzymatic activity of horse spleen apoferritin in iron(II) oxidation was examined using microelectrode oximetry. The reaction exhibits saturation kinetics with respect to both Fe$\sp{2+}$ and O$\sb2$. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe$\sp{2+}$ oxidation occurs in 1-electron steps. At increments of 50 Fe$\sp{2+}$/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. The results of these studies emphasize the role of the protein shell in all phases of core growth.

A …

A Study Of Deoxyribooligonucleotide Duplexes Containing An Unpaired Base Or Mismatched Base Pairs Using: (I) Molecular Modeling, And, (Ii) Proton Exchange Measured With Nuclear Magnetic Resonance Spectroscopy., Bonnie Mary Gunn-Tolson

LSU Historical Dissertations and Theses

Mismatched base pairs have been implicated in mutations and arise from transitions or transversions of the base pairs during replication. An oligonucleotide duplex containing adjacent G$\cdot$A and A$\cdot$A mismatches, d(GCGAAT)$\cdot$d(ATAAGC), has been investigated using molecular mechanics and dynamics. Experimental NMR data obtained for a related molecule was used to constrain the model which suggested an unusual hydrogen-bonding for both mismatches. Between the G$\cdot$A and A$\cdot$A mismatches, defined by the unusual base pairing, there is a large twist angle. The remainder of the base pairs are B-DNA conformations. The effect of an unpaired base on the local geometry of an oligonucleotide …

The Significance Of Beta-72 Asparagine Methylation In C-Phycocyanin., Beth Ann Thomas

LSU Historical Dissertations and Theses

A novel post-translationally modified asparagine, $\gamma$-N-methylasparagine (NMA), has been characterized at the $\beta$-72 site in many phycobiliproteins. The behavior of a NMA-containing peptide under Edman degradation conditions employed for sequence analysis was examined; NMA can be reproducibly identified in protein sequences by careful attention to repetitive yields and the presence of minor peaks. The effects of asparagine methylation on photosynthetic rates in the cyanobacterium Synechococcus PCC 7942 and two methylase-minus mutants was measured by steady-state oxygen evolution in whole cells. The methylase-minus mutants demonstrated lower rates of electron transfer through Photosystem II under conditions in which phycobilisomes were preferentially illuminated …

Partial Purification And Characterization Of A Naturally-Occurring Inhibitor Of Viral Polyprotein Processing., Shaffina John

LSU Historical Dissertations and Theses

An inhibitor activity that prevents the proteolytic processing of Cowpea Mosaic Virus (CPMV) polyprotein has been discovered in crude wheat germ extracts. CPMV is a comovirus that replicates via the synthesis of two polyproteins which are subsequently cleaved by a viral proteinase to yield the functional viral proteins. Since this scheme of replication is common to many other viral groups, a potential target for antivirals is the proteinase responsible for polyprotein processing. A partial purification of this wheat germ inhibitor activity has been accomplished by classical protein purification methods which include ammonium sulfate precipitation, heat treatment, ion exchange, size exclusion …

Submicron Correlations Of Receptor-Positions On Cell Surfaces, Paul St-Pierre

Digitized Theses

Scanning fluorescence correlation spectroscopy (S-FOS) is introduced as a technique to measure the distribution of receptors on cell surfaces. The receptors are made observable by the binding of a specific ligand that is fluorescently labeled. This technique is based on fluctuation analysis through direct calculation of correlation functions. It uses a confocal microscope configuration to obtain fluorescence records as a function of laser beam position on the surface of cells. The fluorescence records present fluctuations that have a characteristic persistence length equal to the laser beam width. The calculated correlation function decays as the laser beam intensity profile which is …

Genomic Characterization Of Steroid Hormone-Binding Globulins, D Alan Underhill

Digitized Theses

Sex-hormone binding globulin (SHBG) and corticosteroid binding globulin (CBG) are the major plasma transport proteins for sex-steroids and glucocorticoids, respectively, and may impact directly upon steroid hormone bioavailability to target cells. Androgen binding protein (ABP) is closely related to SHBG and is believed to maintain an androgenic environment for sperm maturation. The isolation of SHBG-related cDNAs from human testis and a corresponding transcription unit indicates that SHBG and ABP are encoded by the same gene. In addition, the SHBG/ABP gene gives rise to two novel testicular transcripts through a process of alternative splicing. The putative proteins encoded by these transcripts …

Comparative Studies Of The Effects Of Menogaril And Adriamycin On Cell Morphology And Membrane Function Of Parental And Multidrug Resistant Cells, Stewart E. Daly

Digitized Theses

This project was initiated to obtain a better understanding of the cellular mode of action of the anthracycline drug menogaril. Comparative experiments demonstrated that menogaril was as cytotoxic as adriamycin against parental AUX B1 Chinese hamster ovary cells (CHO), however, multidrug resistant (MDR) CH{dollar}\sp{lcub}\rm R{rcub}{dollar}C5 CHO cells, which are characterized by the overexpression of P-glycoprotein relative to the parental cells, were less resistant to menogaril. When parental and MDR cells were exposed to equitoxic concentrations of both drugs, menogaril accumulated to a higher level, however, both drugs accumulated to a significantly higher level in the MDR cells. Immediately after exposure …

Aspects Of Pulmonary Surfactant Metabolism, Rudolf A. Veldhuizen

Digitized Theses

Pulmonary surfactant stabilizes the lung by reducing the surface tension at the air liquid interface of the alveolus. Phosphatidylcholine represents the principal component of surfactant (70% by weight), other lipids account for 20% of surfactant and the remaining 10% is protein. Four surfactant-associated proteins have been identified, surfactant associated protein (SP) SP-A, SP-B, SP-C and SP-D.;This thesis describes the investigations on the following aspects of surfactant metabolism: (1) phosphatidylcholine synthesis, (2) alterations in surfactant after lung transplantation and (3) mechanisms of alveolar surfactant subtype conversion.;(1) The de novo synthesis of acyl-specific phosphatidylcholine via the acylation of glycerol-3-phosphocholine (GPC) has been …

Studies Of The Behaviour Of Lipophilic Molecules In Model Membranes: Applications To Amphotericin B And Glycosphingolipids, Katherine S. Hamilton

Digitized Theses

Our investigations have focused on two model membrane systems: liposomal amphotericin B and glycosphingolipids as minor components in phospholipid bilayers. Although amphotericin B and glycosphingolipids have very diverse biological functions, studying their respective behaviour and interactions in the context of a lipid bilayer is of prime importance.;The association of amphotericin B with lipid vesicles has proven to be a successful modality of treatment, with an antifungal activity that is closely tied to the physical nature (e.g. vesicle size and lipid composition) of the preparation. Using freeze-fracture electron microscopy, density gradient ultracentrifugation, and differential scanning calorimetry we have characterised the basic …

Structural And Biosynthetic Relationships Between The Beta And Epsilon Subunits Of Escherichia Coli F(1)-Atpase, Helmut Garry Dallmann

Digitized Theses

The zero length crosslink between the inhibitory {dollar}\epsilon{dollar} subunit and one catalytic {dollar}\beta{dollar} subunit of E. coli F{dollar}\sb1{dollar}-ATPase ({dollar}\alpha\sb3\beta\sb3\gamma\delta\epsilon{dollar}), induced by 1-ethyl-3- ((3-dimethylamino)propyl) -carbodiimide (EDC), was determined. A crosslinked CNBr fragment derived from {dollar}\beta{dollar}-{dollar}\epsilon{dollar} was identified by SDS-PAGE. Protein sequence analysis revealed the constituent peptides to be {dollar}\beta{dollar}-Asp-380--Met-431 and {dollar}\epsilon{dollar}-Glu-96--Met-138. {dollar}\beta{dollar}-Glu-381 was absent from cycle 2 indicating that it was one of the crosslinked residues, but no potential crosslinked residue in {dollar}\epsilon{dollar} was identified.;An {dollar}\epsilon{dollar} mutant with methionine replacing Val-112 ({dollar}\epsilon{dollar}V112M) was produced, incorporated into F{dollar}\sb1{dollar}, then crosslinked with EDC. A crosslinked CNBr fragment of {dollar}\beta{dollar}-{dollar}\epsilon{dollar}V112M was shown to contain …

Regulation Of Gene Expression Of Surfactant-Associated Proteins In Developing Rabbit Lung, Jiejing Xu

Digitized Theses

Pulmonary surfactant replacement therapy and antenatal glucocorticoid administration are the two treatment strategies used for Respiratory Distress Syndrome. Because of the important roles surfactant-associated proteins play in surfactant function, understanding the mechanisms involved in regulation of the genes encoding for the proteins in fetal development or upon glucocorticoid treatment is therefore of paramount importance in the overall design of an effective treatment regimen. We attempted in this project to investigate the developmental and glucocorticoid regulation of three surfactant proteins, SP-A, SP-B and SP-C, using the rabbit as a model. The mRNA levels for these three proteins were examined in developing …