School of Medicine Theses and Dissertations
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Item Functions of the Unique N-terminus of a GCN5 Histone Acetylase in Toxoplasma gondii(2007-05-18T13:14:16Z) Bhatti, Micah M.; Sullivan, William J., Jr.; Chan, Edward M.; Queener, Sherry F.; Safa, Ahmad R.; Sinai, Anthony P.; Vasko, Michael, R.GCN5 is a histone acetyltransferase (HAT) that remodels chromatin by acetylating lysine residues of histones. The GCN5 HAT identified in Toxoplasma gondii (TgGCN5) contains a unique N-terminal “extension” that bears no similarity to known proteins and is devoid of known protein motifs. The hypothesis of this thesis is the N-terminal extension is critical to the function of TgGCN5. Three possible roles of the N-terminus were investigated: nuclear localization, protein-protein interactions, and substrate recognition. Subcellular localization was determined via immunocytochemistry using parasites expressing recombinant forms of TgGCN5 fused to a FLAG tag. Initial studies performed with parasites expressing full length FLAG-TgGCN5 were positive for nuclear localization. Without the N-terminal extension (FLAG-ΔNT-TgGCN5) the protein remains cytoplasmic. Additional studies mapped a six amino acid motif (RKRVKR) as the nuclear localization signal (NLS). When RKRVKR is fused to a cytoplasmic protein, it gains access to the nucleus. Furthermore, we have established the NLS interacts with Toxoplasma importin α, a protein involved in nuclear trafficking. Interaction with importin α provides evidence that the TgGCN5 N-terminal extension is involved in mediating protein-protein interactions. In order to identify additional interacting proteins, FLAG affinity purification was performed on parasites expressing full length FLAG-TgGCN5 and FLAG-ΔNT-TgGCN5. Upon comparing the results of the two purifications, proteins captured with only full length TgGCN5 may be interacting with the N-terminal extension. Full length TgGCN5 affinity purification indicates an interaction with histone proteins, two different homologues of Ada2 (adapter protein reported to interact with GCN5 homologues), and several heat shock proteins. With regard to substrate recognition, the N-terminal extension of TgGCN5 is dispensable for the acetylation of non-nucleosomal histones in vitro. However, the lysine acetylated by TgGCN5 is surprisingly unique. Other GCN5 homologues preferentially acetylate lysine 14 in histone H3, but TgGCN5 exclusively acetylates lysine 18 in histone H3 and has no activity on lysine 14. Taken together, these results argue that the N-terminal extension of TgGCN5 is critical for mediating protein-protein interactions, including those responsible for trafficking the HAT to the parasite nucleus but does not appear to be required for the acetylation of non-nucleosomal histones.Item The distribution of genetic markers and Q-band chromosomal heteromorphisms in the Kuwaiti population(1979) Al-Nassar, Khaled EidThe distribution of variants of fourteen genetic and fourteen cytogenetic markers is investigated in samples from two Bedouin tribes, the Ajman (n = 52) and the Suluba (n = 52) and from the general population of Kuwait (n = 89). Typical of the many tribal populations in the Arabian peninsula, both Ajman and Suluba are socially isolated from each other. There is little documentation on the ancestral descent of both tribes. However, oral tradition regards Ajman as a deep-rooted Arabian tribe, while the Suluba is thought by some to have originated from the followers of the Crusade camps. The validity of the history of ancestral descent of both tribes is substantiated by a comparison of the distribution of several genetic markers with those of other peninsular communities, and by genetic distance measures between them, the peninsula Arabs of the South-West, and a European population (Italians). Distance measures were calculated by two different methods. Both genetic and cytogenetic marker data obtained from the three Kuwaiti communities contribute significantly to the sparse genetic information on the peninsular populations, and illustrate the degree of genetic microheterogeniety between these communities which was brought about by some social factors that caused their isolation. Gene flow from the neighboring East African populations is evident from the allelic distribution of certain systems such as the Duffy and the Rhesus. Evidence is presented which supports the speculation regarding the prevalence of K of the Kell system and M and S of the MNSs system in the indigeneous peninsular populations. A new salivary amylase isozyrne, Amy K1, was detected in a subject of probable Asiatic Indian descent sampled in Kuwait. Q-band chromosomal heteromorphisms were scored in the three sampled Kuwaiti communities. There was no statistical significance in the differences in frequencies of these heteromorphisms between the three samples. Genetic distances between the Kuwaiti communities and others from the literature were calculated on the basis of Q-band heteromorphic loci. The distances demonstrate the pitfalls in using absolute frequencies of chromosomal variants scored by different research groups for comparative studies. Large Y chromosome was present at high frequencies in the three Kuwaiti communities and was highest among members of the Ajman tribe. This finding suggests that the prevalance of large Y may be a distinguishing cytogenetic feature of the indigenous peninsular populations. Small Y was present in the sample from the general population, but not detected in either sample from the tribal communities. The differences in frequencies of Y chromosome variants between the three sampled communities was found to be statistically significant. Investigation of the 9qh region with the G-11 technique has shown an absence of inversions, partial and complete, of this heteromorphic region in the sample from the general population of Kuwait.Item The nature and mechanism of B-Cytotoxic action of diabetogenic nitrosoureas(1977) Akpan, Jones OkonThis investigation was initiated to test the hypothesis that streptozotocin (STZ) and N-methylnitrosourea (MNU) damage B-cells of islet of Langerhans by depleting islets pyridine nucleotide content. Islets of Langerhans isolated by the collagenase method from nonnal rats were exposed to STZ rmder various experimental conditions and islets pyridine nucleotide content was temporally correlated with cytotoxicity. Metabolic and insulin secretory function of islets of Langerhans were used as indices of B-cell toxicity. The nitrosoureas (STZ and MNU) exerted time-, dose-, and temperature-dependent suppression of glucose-stimulated insulin secretion. Identical studies indicated that rmlike the nitrosoureas, alloxan was B-cytotoxic at 0°c even in the presence of 16.7 mM glucose. Whereas 2 mM alloxan elicited transient but consistent release of insulin by islets perifused in low glucose (1.7 mM) at 37°c, there was no release of insulin by islets perifused with 5 mM STZ, The effect of 5 mM STZ or of 10 mM MNU can be completely inhibited by the simultaneous presence of nicotinamide, isonicotinamide, picolinamide and other primary amides (e.g. pyrazinamide and benzamide) at concentration of 20 mM. Glucose (16.7 mM), pyridine nucleotides (10 mM) and acid derivative of amides (e.g. nicotinic acid; 20 mM) were ineffective. 2-Deoxyglucose (20 mM) or 3-0-methylglucose (20 mM) offered partial protection. After exposure of islets to STZ (5 mM) or MNU (10 mM), complete reversal of the effect was obtained with nicotinamide (20 mM) or its isomers, with time of exposure not exceeding 30 minutes. Beyond 30 minutes, the reversibility was partial or absent, except in the presence of 0.5 mM phenazine methosulfate (PMS) which induced release of insulin in both normal and nitrosoureas pre-treated islets. Insulin releasing action of PMS was dose-, time- and temperature-related; occurred even in the absence of glucose; was inhihitcd hy epinephrine (10 mM, hut not by mannoheptulose (20 mM); and was not potentiatcd by cyclic AMP (5 mM) or theophylline (10 mM). In the perifusion system, the patterns of response induced by PMS (0,5 mM) was spike-like release reaching a maximum in 5 minutes and declining rapidly to half-maximal value in 10 minutes. Normal islets pre-exposed to PMS was refractory to subsequent glucose (16.7 mM) stimulation. Islets pre-treated with STZ (1-5 mM) metaholized less 14c-glucose than control islets. The order of inhibition hy STZ of 14c-glucose metabolism by islet was: l-14C->U- 14C->6-14C-glucose. PMS (0.5 mM) augmented the metabolism of U-14C- and 1-14C glucose by STZ pre-treated islets. However, the metabolism of 6-14C-glucose was unelevated by PMS. The level of NADP+ + NADPII but not the level of NAD, decreased after two minutes exposure of islets to STZ. At thirty minutes, however, the levels of NAD,6-phosphogluconate and NADP+ + NADPII were decreased. The depletion paralleled suppression of insulin secretion. The level of NADP+ + NADPH in islets was decreased more than the level of NAD. Whereas PMS (0.5 mM) elevated the level of NADP+ + NADPH, the level of NAD was not augmented. Islets isolated from rats 3 or more hours after pre-injection with STZ (65 mg/kg) or 6-aminonicotinamide (40 mg/kg) failed to secrete insulin in response to glucose stimulation. However, insulin secretion by such islets was elevated in the presence of PMS (0.5 mM). Whereas PMS induced insulin secretion was much reduced 48 hours after preinjection of rats with STZ, the secretory activity in islets of rats 48 hours after 6-aminonicotinamide injection was slightly decreased. Pyridine nucleotides augmented secretion only in islets of 6-aminonicotinamide pre-injected rats. It is concluded that the immediate response of islets to nitrosoureas in vitro differs from islets response to alloxan. The actions of STZ and MNU are qualitatively similar. The B-cytotoxic effect of nitrosoureas is exerted directly or indirectly on the metabolic and energy coupling functions of pyridine coenzymes. Such an effect could be reversed by supplying the islets with reactive proton donors as substitutes for pyridine nucleotides.Item The effects of 3-mercaptopropionic acid on the cardiovascular system and the content of GABA in specific areas of the brain: further evidence for GABAergic involvement in central cardiovascular control(1984) Alsip, Nancy L.A role has been proposed for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in central cardiovascular control. This proposa 1 was based on the cardiovascular effects of agents which block or mimic the action of GABA on the post-synaptic membrane. This dissertation reported the cardiovascular effects of 3-mercaptopropionic acid (3-MP), an agent which inhibits GABA biosynthesis in the pre-synaptic nerve terminal in anesthetized guinea pigs. 3-MP interrupts GABAergic transmission by decreasing the amount of GABA in the brain. The effect of 3-MP on mean arterial pressure, heart rate and barorflex-induced bradycardia was determined as well as the mechanism(s) involved in observed changes. The content of GABA in four regions of the brain (hypothalamus, medulla, cerebellum and occipital cortex) was determined at the end of each experiment. In anesthetized guinea pigs, 3-MP (195 mg/kg, i.p.) elicited a biphasic response (Type I) in the majority of animals. This response consisted of sympathetically-mediated hypertension and tachycardia superseded by vagally-mediated bradycardia. The other response (Type II) consisted of only the sympathetically-mediated effects. The Type II response was associated with animals in which the vagus nerves were functionally impaired. In all brain regions measured, the GABA levels of both Types I and II were significantly lower than those of control animals. The sympathetically-mediated effects of 3-MP were reversed by chlordiazepoxide, a GABA-facilitory agent. Therefore, the 3-MP-induced cardiovascular effects appeared to reflect GABAergic activity in the bra in which resulted from a reduction of GABA content. The two phases of the Type I response may have resulted from a reduction of GABA content in specific brain regions. A reduction in hypothalamic GABA levels appeared to be related to the sympathetic activation and a reduction in medullary GABA levels appeared to be related to the vagal activation. In unparalyzed animals, 3-MP elicited convulsive movements as well as the described effects on the cardiovascular system. The centrally acting anticonvulsant phenytoin stopped 3-MP-induced motor manifestations of seizure activity without altering either blood pressure or heart rate. Therefore, the cardiovascular effects of 3-MP appeared to occur independent of the convulsive effect of this agent. These results support the hypothesis of GABAergic involvement in the central control of autonomic outflow to the cardiovascular system. The inability of phenytoin to reverse the cardiovascular effects of 3-MP suggests that these effects were independent of 3-MP-induced seizures.Item Studies on the regulation of rat renal gluconeogensis: mechanism of action of somatostatin(1984) Alkhawajah, Abdulaziz MansourLiver is considered the main glucostatic organ in the mammals. However, under a variety of physiological and pathological conditions the kidney plays a significant role in controlling glucose homeostasis. Regulation of renal gluconeogenesis by peptide hormones has not been extensively studied. Somatostatin, the growth hormone release inhibiting factor, has been shown to stimulate renal gluconeogenesis. In this study the detail mechanism of somatostatinstimulated renal glucose production is investigated. Prior treatment of the animals with reserpine to deplete tissues catecholamine stores did not abolish the stimulatory effact of soma tost a tin indicating that catecholamine rel ease may not mediate the enhanced gluconeogenic activity. Somatostatin effect was blocked by the alpha1-antagonist, prazocin, but not by the alpha2 antagonist, yohimbine, suggesting that alpha1 adrenergic receptors may be involved in somatostatin action. somatostatin decreased glucagonstimulated cyclic AMP accumulation and caused small but significant increase in renal cyclic AMP levels. It is proposed that somatostatin may act as a partial agonist to stimulate cyclic AMP production in rat renal tissues. Somatostatin-stimulated renal glucose synthesis is calcium dependent, since in a calcium-free system, somatostatin had no effect. Furthermore, somatostatin increased 45cCa++_ influx into renal tissues. The key rate limiting gluconeogenie reactions is stimulated by somatostatin in the renal cells as demonstrated by the increase in incorporation of [14c] from [14c]-pyruvate and [14c]-bicarbonate into glucose. In addition, somatostatin infusion increased the activities of the key enzymes, phosphoenol pyruvate carboxykinase and pyruvate carboxylase with no effect on fructose 1,6-bisphosphatase nor glucose-6-phosphatase. Somatostatin is shown to bind to one class of recognition sites in the rat renal plasma membranes which showed high Na-K ATPase and adenylate cylase (positive marker enzymes) activites and low activities for succinic dehydrogenase, glucose-6-phosphatase and beta-glucuronidase (negative marker enzymes). The dissociation constant (Kd) for the binding was 0.91 ± 0.06 nM and the binding capacity (Bmax) was 37.59 ± 1.04 fmole/mg protein. Somatostatin and Tyr1-somatostatin displaced [125I]-Tyr1-somatostatin binding with inhibition constant (Kr) values of 31.5 and 100 pM, respectively. This indicates the presence of specific receptors for somatostatin on rat renal cells.Item Item Consequences of linoleic acid deficiency in respect to fatty acid biosynthesis(1964) Allmann, David WilliamItem