Nowadays, computers play an indispensable role in the determination of metal-ligand formation constants and in their application to various situations of analytical, industrial or biological interest. The development of programs and simulation techniques to meet some current problems in bioinorganic chemistry constitutes the broad objective of the present research. Consideration is given to the thermodynamic calculation of complex species concentrations in biological fluids. New methods of solving the mathematical relationships for metal-ligand solution equilibria, particularly in the simulation of large multicomponent systems, are investigated. The ways in which computer simulations are involved in the determination of formation constants are discussed. Principles are developed and applied to problems concerning (i) the calibration of glass electrodes and (ii) the choice of complex species to describe metal-ligand systems under experimental investigation. The function of transition elements in biological systems is briefly reviewed. Emphasis is given to the significance of low-molecular-weight complexes and how a knowledge of their in vivo behaviour can affect bioinorganic drug design. The relationship between copper and rheumatoid arthritis and the importance of equilibria in the regulation of iron metabolism are treated in some detail. New simulation techniques are developed for blood plasma. The results successfully rationalise many bioinorganic phenomena. In particular, the relative ability of a series of chelating agents to compete with proteins for metal ions in plasma is correlated with the urinary excretion of trace elements that they cause. Further simulations extend the approach to other biofluids and to medical solutions intended for intravenous infusion.

Call number: LD2668 .T4 1978 S85 / Master of Science

This research is directed at developing the interplay of experimental and computational methods in the area of biochemical mass spectrometry. The experimental method is that of electrospray ionization mass spectrometry (ESI-MS). The computational methods employed are those of semi-empirical quantum mechanics and molecular modeling.The use of Electrospray Mass Spectrometry was developed to investigate whole proteins and the non-covalent complexes that may be formed with small molecules. This method provides the soft ionization needed to accurately determine a noncovalently bound complex's mass with an error of less than 0.1 %. An original design electrospray ionization source (ESI) and a syringe pump have been built to fulfill the goals of the research. The ESI source design has been published in The Journal of the American Society for Mass Spectrometry, (1993, 4, 964-967).In this work the protein selected was cytochrome c and its variants from different species. The small molecules chosen were a broad class of structures known as crown ethers. With the ESI technique the proteins are prepared in an acidic solution that fully protonates the solvent-exposed basic amino acid residues. This provides the protein with many positive charges which makes the analysis by ESI mass spectrometry possible with a single quadrupole instrument, an Extrel ELQ 400. The mass of the protein is divided by the number of positive charges. The small molecules, the crown ethers, were chosen due to their ability to bind ammonium ion and protonated amino groups. This binding is non-covalent, hydrogen bonds stabilize the complex formation. Because this complex is non-covalent in nature the charge of proteins does not change. To aid in the interpretation of our electrospray mass spectra we have originated a new kind of linear plot for use with ESI data. It was found that in using the ESI technique that ion currents representing non-covalent complexes of cytochrome c and crown ethers could be observed in the mass spectra. The measurements of the total ion counts of peaks in the mass spectra allowed binding constants to be calculated. This had not been reported before in the literature.The accurate weight determination and the characteristic charge distribution in the ESI mass spectrum provides data suitable for computer modeling. The nature of the protein's positive charges in ESI had not been well defined. The experimentally determined binding constants allowed comparison to results from computational chemistry and molecular modeling. This result shows that the binding occurs at specific, protonated amino acid residues. The calculations performed yielding the Heats of Formation (OH f) for protonated amino acids complexed with crown ethers indicates that the OH binding of crowns increases from histidine, to arginine, to lysine. The use of a 3D model of cytochrome c from crystallographic data provided in the Brookhaven Protein Database and the SYBYL molecular modeling program allows a structural correlation to be made between the 3D model of the protein and protein/crown ether complex. The stoichiometric ratios of bound crown ether to protein determined from experiment, along with the computational results, have been used to rationalize a protein molecular model that allows predictions to be made about the potential for binding of other small molecules. / Department of Chemistry

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

At the beginning of this study, the granule localisation and regulation of release of human neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and type TV/V collagenases, respectively), enzymes highly active against the extracellular matrix (ECM) and thought to be relevant in invasion and inflammation, had been established while that of their inhibitor, tissue inhibitor of matrix metalloproteinases-I (TIMP-1), had not. Electron microscopy immunogold labelling of cryoultramicrotomy sections for granule marker proteins, lysosome-associated membrane proteins (LAMPs) and endocytosed bovine serum albumin-coated gold probes, followed by stereology, established that TIMP-1 was mainly located in a distinct oval, electron translucent organelle, a little larger than azurophil granules. A lack of labelling for endocytic markers and for glycosylphosphatidylinositol-anchored proteins, established using granule fractionation and immunolabelling to be markers for the secretory vesicles, and LAMPs-1 and -2, indicated the non-endosomal, non-secretory and nonlysosomal nature of this organelle. Density gradient cofractionation with the least dense secretory vesicle population and some pleiomorphism of the organelle suggested that it is a "vesicle" rather than a "granule" population. Colocalisation with proMMP-9 in minor subpopulations suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and termination differentiation, but before secretory vesicle synthesis. Immunolabelling of phagocytosed and pulse-chased IgG-opsonised latex beads showed that specific and azurophil granules and a small number of proMMP-8-containing granules (a specific granule subpopulation) fuse with the phagosome whereas the TIMP-1 vesicle and proMMP-9-containing granules do not, suggesting that the latter play no role in phagosomal destruction of IgG-opsonised bacteria and that their phagosomal release is not calcium regulated. However, studies using the calcium ionophore, ionomycin, and monitoring extracellular granule marker protein release upon addition of increasing levels of extracellular calcium, showed that all granules, except the TIMP-1 vesicle, appeared to be calcium regulated. This suggests that the regulation of proMMP-9 release is not exclusively via calcium and that TIMP-1 vesicle release is not calcium regulated. Whereas most granules were shown to be associated with microtubule-like structures, the TIMP-1 vesicle and proMMP-9-containing granules were shown to associate with two morphologically different cytoskeletal elements, neither resembling actin nor tubulin. These elements, and the release of the TIMP-1 vesicle and proMMP-9-containing granules, need to be studied further, but results achieved to date may explain the observed differential mode of release of TIMP-1 relative to proMMP-9. The proMMP-9-binding and inhibitory capacity of a 66 kDa high molecular mass form of TIMP-1 was demonstrated in PMNL homogenates and plasma using western ligand blots and a novel reverse zymography method. The role and relevance of this form remains unknown as does the relevance and potential role of proMMP-9ffIMP-1 complexes seen during isolation procedures. The proMMP-9ffIMP-1 complex may occur in vivo, as evidenced by immunolocalisation studies, and, together with TIMP-1 released from its own discrete vesicle population, may be responsible for the fine regulation of extracellular proteolysis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Indiana University-Purdue University Indianapolis (IUPUI) / The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.

Indiana University-Purdue University Indianapolis (IUPUI) / The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.