No description available.

No description available.

Covert stroke (CS) comprises lesions in the brain often associated by risk factors such as a diet high in fat, salt, cholesterol and sugar (HFSCS). Developing a rodent model for CS incorporating these characteristics is useful for developing and testing interventions. The purpose of this thesis was to determine if magnetic resonance (MR) can detect brain abnormalities to confirm this model will have the desired anatomical effects. Ex vivo MR showed brain abnormalities for rats with the induced lesions and fed the HFSCS diet. Spectra acquired on the fixed livers had an average percent area under the fat peak relative to the water peak of (20±4)% for HFSCS and (2±2)% for control. In vivo MR images had significant differences between surgeries to induce the lesions (p=0.04). These results show that applying MR identified abnormalities in the rat model and therefore is important in the development of this CS rodent model.

No description available.

Proton nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the interaction of yeast phosphoglycerate kinase (PGK) with its phosphate containing substrates, ATP and 3-phosphoglycerate (3-PG). The application of one-dimensional and, for the first time, two-dimensional proton NMR techniques to this large protein has enabled specific resonance assignments to be made. Assignment has been aided by the investigation of specifically deuterated protein and site-specific mutant forms of the protein, including the isolated N- and C-domains. The effects of ATP and 3-PG binding on the proton NMR spectrum of yeast PGK have been characterised and the assigned resonances used as local probes of structural and dynamic changes. Two binding sites have been determined for the nucleotide substrate, ATP, the occupancies of which are dependent on Mg²⁺ concentration. One site corresponds to the catalytic site determined crystallographically. A single binding site was found for 3-PG. This binding was shown to cause highly specific conformational changes throughout the N-domain and the interdomain region, which involve the relative movement of at least three α-helices. Investigation of 3-PG binding to several site-specific mutant forms of yeast PGK revealed a critical role for arginine 168 in the propagation of these changes. The general binding of anions to yeast PGK was investigated using the paramagnetic probes [Cr(CN)₆]^3- and [Fe(CN)₆]^3-, and the diamagnetic anion [Co(CN)₆]^3-. The primary anion binding site was determined from [Cr(CN)₆]^3- broadening data and found to share some side-chains involved in 3-PG binding, namely histidine 62 and arginine 168. Evidence for a secondary anion site was found. The anion binding data is discussed in view of the complex activation/inhibition effects of anions on the catalytic activity. Investigation of the isolated N- and C-domains showed that both can fold independently and confirmed that the C-domain is a nucleotide binding domain. It appears that the presence of the interdomain residues and/or the C-terminal peptide are necessary for 3-PG binding to the N-domain. This work shows that the specificity of the substrates is in binding, as expected, but also in the motions induced in the protein as a whole.

Covert stroke (CS) comprises lesions in the brain often associated by risk factors such as a diet high in fat, salt, cholesterol and sugar (HFSCS). Developing a rodent model for CS incorporating these characteristics is useful for developing and testing interventions. The purpose of this thesis was to determine if magnetic resonance (MR) can detect brain abnormalities to confirm this model will have the desired anatomical effects. Ex vivo MR showed brain abnormalities for rats with the induced lesions and fed the HFSCS diet. Spectra acquired on the fixed livers had an average percent area under the fat peak relative to the water peak of (20±4)% for HFSCS and (2±2)% for control. In vivo MR images had significant differences between surgeries to induce the lesions (p=0.04). These results show that applying MR identified abnormalities in the rat model and therefore is important in the development of this CS rodent model.

The calcium binding epidermal growth factor-like (cbEGF) 12-13 domain pair from human fibrillin-1 was the focus of studies for this dissertation. Various nuclear magnetic resonance (NMR) spectroscopy techniques were employed to analyse the calcium binding, structural and dynamic properties of this pair, and to assess the effects of a disease-causing mutation. Fibrillin-1 is a mosaic protein composed mainly of 43 cbEGF domains arranged as multiple, tandem repeats, and mutations within fibrillin-1 have been linked to Marfan syndrome (MFS). 66% of MFS-causing mutations identified thus far are localised to cbEGF domains, emphasising that the native properties of these domains are critical to the functional integrity of this protein. The cbEGF 12-13 pair is found within the longest run of cbEGFs in fibrillin-1, and many mutations that cluster in this region are associated with the severe, neonatal form of MFS. It is thought that this region may be important for fibrillin-1 assembly into 10- 12nm connective tissue microfibrils. Calcium binding studies of cbEGF 12-13 demonstrated that cbEGF 13 contains the highest affinity site thus far investigated from human fibrillin-1. Comparison with previous results showed that fibrillin-1 cbEGF calcium binding affinity can be significantly modulated by the type of domain which is linked to its N-terminus, and also highlighted the high affinity of the "neonatal" region. The NMR solution structure of cbEGF 12-13 is a near-linear, rod-like arrangement of two cbEGF domains, with both exhibiting secondary structure characteristic of this domain type. The rod-like arrangement is stabilised by calcium binding by cbEGF 13 and by hydrophobic interdomain packing interactions. This observation supports the hypothesis that all Class I EGF/cbEGF-cbEGF pairs, characterised by a single linker residue, possess this rod-like structure. The structure also exhibits additional packing interactions to those previously observed for cbEGF32- 33 from fibrillin-1, which may explain the higher calcium binding affinity of cbEGF13. A model of cbEGF 11-15, created based on structural data for cbEGF 12-13 and a model of cbEGF32-36, has highlighted a potential protein binding interface, which encompasses all known neonatal MFS mutations, as well as a flexible, unstructured loop region of cbEGF 12. Backbone dynamics data confirmed the extended structure of cbEGF 12-13. These data, combined with previous data for cbEGF32-33, highlighted a potential dynamics signature for Class I cbEGF domain pairs. Comparison of data for these pairs also suggested that, in addition to the role of calcium in stabilising rigidity on the picoto millisecond time-scale, calcium affinity may play a key role in determining the anisotropy of cbEGF pairs. Possible dynamic explanations for the variation in calcium binding affinity of cbEGF domains from human fibrillin-1 were also noted. The Gl 127S mutation located in cbEGF 13 of fibrillin-1 causes a mild variant of MFS. NMR studies of the G1127S cbEGF12-13 mutant pair showed that cbEGF12 may chaperone folding of mutant cbEGF 13, an effect most likely mediated through interdomain packing interactions. These studies have also shown that the effects of this mutation are localised to cbEGF13, suggesting that a "partial" MFS phenotype is the result of altered structural, dynamic and/or calcium binding properties of this domain.

Most cellular magnesium is bound, yet it is the concentration of free magnesium, [Mg²⁺]_free, in red blood cells that is vital in the regulation of enzyme activity and ion transport. It is unknown how changes in total blood magnesium affect the [Mg²⁺]_free within red blood cells or in tissue, because the presence of other cations, especially H⁺ and potassium, K⁺ , affects the degree to which Mg²⁺ is bound. Consequently, this Thesis presents a new ³¹P NMR spectroscopic method to measure [Mg²⁺]_free in blood, which analyses the changes in the phosphorus chemical shifts of ATP and 2,3-DPG using theoretical equations expressing the observed chemical shift as a function of pH, K⁺ and [Mg²⁺]_free, over the pH range of 5.75 to 8.5 and [Mg²⁺]_free range 0 to 5 mM. The equations were adjusted for the binding of haemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K⁺ and Hb. These equations enabled, for the first time, the simultaneous analyses of the chemical shifts of 3P-DPG and β-ATP to measure both intracellular 04- pH and [Mg²⁺]_free in normal and sickle blood. To simulate in vivo 100% oxygenated blood, samples were prepared for analysis by equilibration with a mixture of O₂ and CO₂, adjusted to give a pCO₂ of 40 mmHg and pO₂ > 150 mmHg. Under these conditions, normal whole blood had an intracellular pH of 7.20 ± 0.02 and a [Mg²⁺]_free of 0.41 ± 0.03 mM (n = 33). Further work determined blood pH and [Mg²⁺]_free for several clinical conditions including sickle cell anaemia, pre-eclampsia, hypoxia, patients with sub-arachnoid haemorrhage and chronic fatigue syndrome. This Thesis has demonstrated the potential of this new technique to evaluate the importance of [Mg²⁺]_free in the regulation of metabolite concentration and metabolic function, and to elucidate some of the properties of magnesium transport across the erythrocyte cell membrane.

Some physicochemical properties of several series of transition metal chalcocarbonyls such as ((eta)('6)-C(,6)H(,6))Cr(CO)(,2)(CX) and Cr(CO)(,5)(CX) (X = O, S, Se) have been investigated. In particular, the infrared, ('13)C and ('17)O nuclear magnetic resonance and ultraviolet spectra have been examined. The results show that the order of (sigma)-donor and (pi)-acceptor capabilities of the ligands is CO CS. The M((pi)) (--->) CX((pi)*) ultraviolet charge-transfer bands are shown to correlate with the respective (mu)'(,MCX) and ('13)C NMR data. Also, the ('13)C NMR chemical shifts and GQVFF force constants are found to be highly correlated. The ('17)O NMR spectra of the metal chalcocarbonyl complexes display chemical shifts in the opposite direction to the corresponding ('13)C ones. No correlation is found between the ('17)O shieldings and the other spectroscopic data. Reaction of Cr(CO)(,5)(CX) (X = S, Se) with halide ions (Y('-)) afforded mixtures of {Cr(CO)(,5)Y}('-) and trans-{Cr(CO)(,4)(CX)Y}('-) while Cr(CO)(,5)(CS) reacted with cyclohexylamine to give Cr(CO)(,5)(CNC(,6)H(,11)). The similarities and differences in the physicochemical behaviour of the metal chalcocarbonyls compared to related systems are discussed in the light of the different bonding patterns.

Includes bibliographies / x, 149 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1986