Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes used / Physics / M. Sc. (Physics)

The primary goal of my thesis research was to characterize the basis for the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in primary culture diabetic cardiomyocytes. Toward this goal, I have investigated several key regulatory sites in this signaling pathway which could promote the hypersensitive activation of phosphorylase. Specifically, I investigated (1) which adrenergic receptors are involved in mediating the hypersensitive response of glycogen phosphorylase to epinephrine stimulation; (2) whether the presence of fatty acid metabolites affects phosphorylase activation; (3) whether the hypersensitive response of phosphorylase results from altered signal transduction through the β-adrenergic receptor system or from a post-receptor defect; and (4) the potential role for phosphorylase kinase in mediating the hypersensitive response of phosphorylase to catecholamine stimulation. The basis for adrenergic receptor mediation of the catecholamine-induced activation of glycogen phosphorylase was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. Cells derived from diabetic animals exhibited a hypersensitive response to epinephrine stimulation which was apparent 3 hours after cell isolation and was further enhanced upon maintenance of the myocytes in culture for 24 hours. Normal cells initially lacked the hypersensitive response to epinephrine stimulation although upon maintenance of these cells in culture for 24 hours, the hypersensitive response was acquired in vitro. To assess alpha- and beta- adrenergic mediation of the response, normal and diabetic cardiomyocytes were incubated with propranolol, a β-receptor antagonist, prior to direct α1receptor stimulation with phenylephrine. Both normal and diabetic myocytes failed to undergo activation of phosphorylase in 3 or 24 hour cell cultures. In addition, the effects of epinephrine on phosphorylase activation were completely inhibited by propranolol whereas prazosin, an α-receptor antagonist, was unsuccessful. This data suggests that the hypersensitive response of glycogen phosphorylase in normal and diabetic cardiomyocytes is solely mediated through β-adrenergic receptor activation. Since the accumulation of various fatty acid metabolites can affect certain enzymes and signal transduction pathways within the cell, the potential effect of various fatty acid metabolites on phosphorylase activation was investigated. To determine the potential effects of fatty acid metabolites on phosphorylase activation in cultured cardiomyocytes, normal and alloxan-diabetic cells were incubated with either carnitine or palmitoylcarnitine prior to stimulation with epinephrine. Pretreatment of cardiomyocytes with or without carnitine or palmitoylcarnitine for 3 or 24 hours before epinephrine stimulation failed to alter phosphorylase activation. The addition of exogenous carnitine in the absence and presence of insulin was also unsuccessful in attenuating the hypersensitive phosphorylase activation response in 3 and 24 hour, normal and alloxan-diabetic derived cardiomyocytes. To determine if carnitine palmitoyltransferase 1 (CPT-1) activity was responsible for the hypersensitive response of phosphorylase in the diabetic myocytes, both normal and diabetic myocytes were maintained for 3 and 24 hours in the absence and presence of etomoxir, a CPT-1 inhibitor. Subsequent activation of phosphorylase by epinephrine in normal and diabetic myocytes was unaltered in the presence of etomoxir. Collectively, these data fail to support a critical role for fatty acid metabolite involvement in the hypersensitive activation of glycogen phosphorylase in acute, alloxan-diabetic cardiomyocytes. To assess potential G-protein involvement in the response, normal and diabetic-derived myocytes were incubated with either cholera or pertussis toxin prior to hormonal stimulation. Pretreatment of cardiomyocytes with cholera toxin resulted in a potentiated response to epinephrine stimulation whereas pertussis toxin did not affect the activation of this signaling pathway. To determine if the enhanced response of phosphorylase activation resulted from an alteration in adenylyl cyclase activation, the cells were challenged with forskolin. After 3 hours in primary culture, diabetic cardiomyocytes exhibited a hypersensitive response to forskolin stimulation relative to normal cells. However, after 24 hours in culture, both normal and diabetic myocytes responded identically to forskolin challenge. The present data suggest that a cholera toxin sensitive G-protein mediates the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in diabetic cardiomyocytes. This response, which is present in alloxan-diabetic cells, and is induced in vitroin normal cardiomyocytes, is primarily due to a defect at a post-receptor site. To assess the role of phosphorylase kinase in the hypersensitive activation of glycogen phosphorylase in the diabetic heart, phosphorylase kinase activity was measured initially in perfused hearts (to optimize the assay parameters) and subsequently in primary culture cardiomyocytes. Results from these experiments demonstrate that the present method for measuring phosphorylase kinase activity is a reliable indicator of the enzyme's activity in the heart, although the assay conditions must be further optimized before this system can be applied to the measurement of phosphorylase kinase activity in primary cultured cardiomyocytes.

Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes used / Physics / M. Sc. (Physics)

Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.

No / Recent studies have revealed extensive cortical demyelination in patients with progressive multiple sclerosis (MS). Demyelination in gray matter lesions is associated with activation of microglia. Macrophages and microglia are known to express myeloperoxidase (MPO) and generate reactive oxygen species during myelin phagocytosis in the white matter. In the present study we examined the extent of microglial activation in the cerebral cortex and the relationship of microglial activation and MPO activity to cortical demyelination. Twenty-one cases of neuropathologically confirmed multiple sclerosis, with 34 cortical lesions, were used to assess microglial activation. HLA-DR immunolabeling of activated microglia was significantly higher in demyelinated MS cortex than control cortex and, within the MS cohort, was significantly greater within cortical lesions than in matched non-demyelinated areas of cortex. In homogenates of MS cortex, cortical demyelination was associated with significantly elevated MPO activity. Immunohistochemistry revealed MPO in CD68-positive microglia within cortical plaques, particularly toward the edge of the plaques, but not in microglia in adjacent non-demyelinated cortex. Cortical demyelination in MS is associated with increased activity of MPO, which is expressed by a CD68-positive subset of activated microglia, suggesting that microglial production of reactive oxygen species is likely to be involved in cortical demyelination.

No / Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet function at sites of vascular injury. Here we show that thrombospondin-1 (TSP-1) prevents cAMP/protein kinase A (PKA) signaling through a CD36-dependent mechanism. Prostaglandin E(1) (PGE(1)) induced a robust inhibition of both platelet aggregation and platelet arrest under physiologic conditions of flow. Exogenous TSP-1 reduced significantly PGE(1)-mediated inhibition of both platelet aggregation and platelet arrest. TSP-1 prevented PGE(1)-stimulated cAMP accrual and phosphorylation of PKA substrates, through a mechanism requiring phosphodiesterase3A. TSP-1 also inhibited VASP phosphorylation stimulated by the nonhydrolyzable cAMP analog, 8-bromo-cAMP, indicating that it may regulate cAMP-mediated activation of PKA. The inhibitory effect of TSP-1 on cAMP signaling could be reproduced with a peptide possessing a CD36 binding sequence of TSP-1, while the effects of TSP-1 were prevented by a CD36 blocking antibody. TSP-1 and the CD36 binding peptide induced phosphorylation of Src kinases, p38 and JNK. Moreover, inhibition of Src kinases blocked TSP-1-mediated regulation of cAMP concentrations and the phosphorylation of VASP, indicating that TSP-1 modulated the cAMP/PKA signaling events through a tyrosine kinase-dependent pathway downstream of CD36. These data reveal a new role for TSP-1 in promoting platelet aggregation through modulation of the cAMP-PKA signaling pathway.

No / Fibroblast growth factor receptors (FGFR) play key roles in proliferation, differentiation, and tumorigenesis. Many urothelial carcinomas contain activating point mutations or increased expression of FGFR3. However, little is known about the role of other FGFRs. We examined FGFR expression in telomerase-immortalized normal human urothelial cells, urothelial carcinoma cell lines, and tumor samples and showed that FGFR1 expression is increased in a high proportion of cell lines and tumors independent of stage and grade. To determine the role of FGFR1 in low-stage bladder cancer, we overexpressed FGFR1 in telomerase-immortalized normal human urothelial cells and examined changes in proliferation and cell survival in response to FGF2. FGFR1 stimulation increased proliferation and reduced apoptosis. To elucidate the mechanistic basis for these alterations, we examined the signaling cascades activated by FGFR1. FRS2alpha and PLCgamma were activated in response to FGF2, leading to activation of the mitogen-activated protein kinase pathway. The level of mitogen-activated protein kinase activation correlated with the level of cyclin D1, MCL1, and phospho-BAD, which also correlated with FGFR-induced proliferation and survival. Knockdown of FGFR1 in urothelial carcinoma cell lines revealed differential FGFR1 dependence. JMSU1 cells were dependent on FGFR1 expression for survival but three other cell lines were not. Two cell lines (JMSU1 and UMUC3) were dependent on FGFR1 for growth in soft agar. Only one of the cell lines tested (UMUC3) was frankly tumorigenic; here, FGFR1 knockdown inhibited tumor growth. Our results indicate that FGFR1 has significant effects on urothelial cell phenotype and may represent a useful therapeutic target in some cases of urothelial carcinoma.