<p>With the advent of radioligand binding assays, central nervous system dopamine receptors have been well characterized in their membrane bound state. These receptors have been grouped into D-1 and D-2 subclasses on the basis of their relationship to the enzyme adenylate cyclase and affinities for dopamine agonists and antagonists. The dopamine D-2 receptor is considered relevant to the behavioral and pharmacological effects of neuroleptic drugs. The studies presented in this dissertation describe a successful method of solubilization of bovine striatal membrane bound dopamine D-2 receptor. The solubilized receptor exhibited typical pharmacological characteristics to that of membrane bound dopamine D-2 receptor. The rank order potency of agonists and antagonists to displace [³H]spiroperidol binding was the same as those observed with the membrane bound receptor. Analysis of the [³H]spiroperidol/agonist competition curves and the [³H]NPA binding revealed the retention of high and low affinity states of dopamine D-2 receptor in the solubilized preparation. This study demonstrated for the first time, a successful affinity chromatography method for the purification of dopamine D-2 receptor. One cycle affinity purification resulted in a 2000-fold enrichment of dopamine D-2 receptor activity with a recovery of 12% from the membrane-bound state and a specific activity of 169,600 fmol/mg protein (assayed with [³H]spiroperidol). The order of potency of D-2 agonists (N-propylnorapomorphine > N0434 > apomorphine > dopamine) and antagonists (spiroperidol > (+)-butaclamol > domperidone) with a purified preparation was found to be similar to that of the membrane bond or solubilized dopamine D-2 receptor. The adsorption of receptor to the affinity matrix was biospecific as pre-incubation of the solubilized preparation with D-2 receptor agonists or antagonists blocked retention of receptor activity. Elution of receptor was also biospecific as dopaminergic drugs were effective in eluting the bound receptor. Affinity purified preparations should be useful in producing monoclonal antibody to dopamine D-2 receptor and also prove to be important in understanding the molecular events from receptor drug binding to final response.</p> / Doctor of Philosophy (PhD)

<p>A. The involvement of central catecholaminergic neurons in tonic gonadotrophin release in male rats was investigated.</p> <p>(i) Animals were given a single intraventricular injection of 6-hydroxydopamine hydrochloride (6-ODHA; 170 μg free base) dissolved in 0.001 N HCl. Plasma LH and FSH were measured by double antibody radioimmunoassay. LH in the 6-OHDA treated animals was significantly reduced after one hour and remained consistently lower for eight hours. The controls, after a transient elevation, showed no significant change. No difference in LH concentrations was found between experimental groups sampled at two days or later. FSH in treated animals showed less consistent difference from the controls.</p> <p>(ii) Animals given an intraventricular injection of 6-OHDA dissolved in different vehicles, with varied pH and osmolarity, gave similar LH and FSH results which suggests the quinone derivative of 6-OHDA is effective in producing the catecholaminergic impairment.</p> <p>(iii) Administration of 6-OHDA dissolved in different vehicles, with varied pH and osmolarity, gave similar LH and FSH results which suggests the quinone derivative of 6-OHDA is effective in producing the catecholaminergic impairment.</p> <p>These findings indicate the LH release in the male rat may be controlled (or at least modulated) by a central adrenergic mechanism.</p> <p>B. The involvement of the ventral noradrenergic ascending pathway in tonic gonadotrophin release was investigated. Bilateral injection of 6-hydroxydopamine hydrochloride given into the preoptic area of the medial forebrain bundle significantly reduced plasma LH at all times tested. Plasma FSH was not significantly different. These results suggest that tonic LH release is modulated by an extrahypothalamic mechanism.</p> <p>C. The hypothalamic catecholamine content was pharmacologically manipulated with various drugs.</p> <p>(i) An intraperitoneal injection of protriptyline was given before 6-OHDA in order to selectively reduce the hypothalamic content of dopamine. Plasma LH, but not FSH was significantly reduced 3 hours after 6-OHDA in animals pretreated with protriptyline compared to controls. Neither plasma nor testicular testosterone were significantly different between groups.</p> <p>(ii) Animals given DL-threo-dihydroxyphenylserine showed a significant increase in plasma LH (previuosly decreased by 6-OHDA) compared to controls. DL-threo-dihydroxyphenylserine selectively restores the noradrenaline content in the hypothalamus.</p> <p>(iii) γ-Butyrolactone blocks the central release of dopamine and produces anaesthesia resembling deep sleep. No consistent significant differences were found in either plasma LH or FSH at any of the times or doses tested in animals given either γ-butyrolactone or a metabolite, γ-hydroxybutyric acid, compared to the control group receiving saline injection alone.</p> <p>(iv) 6-hydroxydopa which selectively reduces brain noradrenaline content, had no effect on plasma LH and FSH.</p> <p>The above findings give further support to the concept of modulation of tonic gonadotrophin release by an extrahypotalamic noradrenergic mechanism.</p> / Doctor of Philosophy (PhD)

<p>Cellular transformation by human adenoviruses requires the expression of two early transcription units, E1A (0 to 4.5% of the genome) and E1B (4.5 to 11.2%). The products of both genes are required to disrupt normal cellular growth control and morphology. Early region 1A products alone can induce cellular immortalization, but establishment of fully transformed cells requires cooperation with E1B. Early region 1B produces two unrelated proteins, 496R and 176R, each of which can act independently with E1A to induce cellular transformation.</p> <p>176R is a membrane associated protein whose molecular role in transformation is not known. Anti-peptide sera specific for the amino- and carboxy-termini of 176R were produced. These two sera were used for further purification and biochemical characterization of the protein. The 176R migrated as two species termed 18.5 and 19K on SDS-PAGE. The generation of the two species was not due to proteolysis, nor any post-translational modification investigated.</p> <p>Two post-translational modifications of 176R were identified: phosphorylation and acylation. The phosphorylation site was mapped to serine residue 164, and a mutant virus in which this amino acid was altered, pm2204, was produced by oligonucleotide-directed mutagenesis. Characterization of pm2204 as well as an E1 plasmid carrying this mutation suggested that phosphorylation is not important for the biological activity of 176R. 176R was also found to contain covalently bound palmitate and myristate. Both tryptic and chymotryptic peptide mapping as well as CNBr cleavage were used to localize the acylation site. These experiments suggested that multiple acylation sites exist, and at least one site was localized to the amino terminal region by tryptic peptide mapping. The linkage of fatty acid to 176R was o found to be via an amide, or an unusually stable ester bond to an internal amino acid. Acylation of 176R likely facilitates its membrane association, but it was not possible to test this hypothesis without precise mapping of the acylation site(s) and construction of specific mutants.</p> / Doctor of Philosophy (PhD)

<p>The aim of the present study was to characterize the pituitary GnRH binding site in the rabbit and investigate its possible role in sexual maturation of the female rabbit. A radioligand binding assay was established and the presence of specific ¹²⁵I-DAla⁶EA binding sites in the anterior pituitary gland of the rabbit was demonstrated. ¹²⁵I-DA1a⁶EA binding was saturable, specific, displaceable, reversible, correlated with increasing tissue concentrations and susceptible to physiological manipulation. Significant ¹²⁵I-DA1a⁶EA binding was not present in the rabbit ovary suggesting that GnRH or GnRH-related peptides are not directly involved in the control of luteal function of the rabbit. 12S1_DAla6EA binding indicated the presence of two binding sites in the female adult rabbit pituitary; a high affinity, low capacity site (Kd= 0.3-0.4 nM; Bmax = 100-200 fmol/mg protein) and a lower affinity, high capacity site (Kd= 30 nM; Bmax=5-8000 fmol/mg protein). Ontogeny of ¹²⁵I-DA1a⁶EA binding in the female rabbit (40 to 120 days of age) did not show a correlation between binding site number and serum LH. In addition, the net serum LH response in female rabbits to a subcutaneous injection of DA1a⁶EA (10ng, 100ng, 1μg per kg body weight) was not significantly different between animals 40, 75 and 120 days of age. This suggests that a decrease in pituitary responsiveness to GnRH is not associated with sexual maturation in the female rabbit. Results indicate that factors other than and/or in addition to GnRH binding site number, such as post-receptor events play a role in gonadotropin secretion in the female rabbit.</p> / Doctor of Philosophy (PhD)

<p>Roberts syndrome (RS) is a rare autosomal recessive developmental disorder that is characterized by severe pre- and post-natal growth retardation, tetraphocomelia and various craniofacial abnormalities. In some patients with Roberts syndrome (RS+) but not others (RS-), RS is associated with a cytogenetic abnormality which presents as an unusual "puffing" of the constitutive heterochromatin and nucleolar organizing regions. The decondensed appearance of the constitutive heterochromatin suggests an alteration in chromatin structure in RS+. Two main questions were addressed in this study; (1) are altered levels of DNA methylation associated with the constitutive heterochromatin abnormality of RS+ chromosomes and (2) is the timing of DNA replication of constitutive heterochromatin, relative to the rest of the genome, altered in RS+ lymphoblast cells? Levels of methylated cytosine residues of RS+ and RS- heterochromatin DNA, assessed using MspI and HpaII isoschizomer restriction enzyme digestion and Southern blot hybridization with a repetitive probe (D15Z1) to the heterochromatin DNA, were found to be significantly lower (p = 0.048) in RS+ fibroblast cell strains compared to control and RS- cell strains. In addition to this, D15Z1 DNA methylation levels differed significantly (p ≤ 0.05) at early and late passage in fibroblast cell strains of all three groups, suggesting that decreased levels of DNA methylation were associated with an in vitro aging effect. The chromosome replication patterns of the constitutive heterochromatin regions of chromosomes 1, 9, 16 and Y were investigated using terminal 5-bromodeoxyuridine labelling and an S phase subclassification system. A significant delay (p < 0.001 for male cell lines; p<0.05 for female cell lines) was observed in the timing of RS+ constitutive heterochromatin. These results suggest that the cytogenetic abnormality of the constitutive heterochromatin of RS+ cells is associated with alterations in DNA conformation and synthesis.</p> / Doctor of Philosophy (PhD)

<p>Th~ thermoregulatory response to exercise in the heat, especia!ly sweating rate, differs between children and adults. This project investigated the effect of physical maturation on the thermoregulatory response to exercise (50% V02mc.x) in the heat (42°C 20%RH) among circum-pubertal boys, using a mixed cross-sectional, longitudinal design. Subjects were initially divided into three groups, based on Tanner (pubic hair) staging: 16 pre-pubenal (PP, Tanner 1), 15 mid-pubertal (MP, Tanner 2,3,4) and 5 Iatepubertal (LP, Tanner 5). The thermoregulatory response was observed every 6 months for a period of 18 months (4 sessions). Thirty of the 36 boys completed the four sessions. During each session, the exercise task :onsisted of three 20-min bouts of cycling and 10 min rest periods. Measurements included rectal and skin temperatures and hean rate continuously, VO:! at the midpoint of the second bout, forearm blood flow (FBF) upon entry to the climatic chamber and immediately following each exercise bout, sweat collection during each bout, photography of sweat drops after bouts 1 and 2, and whole body sweating rate. Blood samples before and after exercise in the heat were analyzed for hormonal, lactate and electrolyte concentrations. Body temperatures tended to be higher among LP and FBF was higher among PP. However, the rate of heat exchange and heat storage per kg body weight were similar among groups. Sweating rate per body surface area and per gland were higher among LP compared to PP. This was accompanied by lower sweat lactate concentrations during the initial stages of exercise, hi~her lactate excretion rate per gland. ~l')wer activated sw~at gland population density, and higher mean sweat drop area. Aldosterone and prolactin concentrations increased during exercise in the heat in all groups. The increase in prolactin was greater among LP compared to PP. Longitudinal observations tended to support cross-sectional findings. It is concluded that, in boys a) heat exchange and heat storage are similar among preto late-pubescents exercising in dry heat; b) physical maturation is characterized by enhanced sweating rate per body surface area and per gland, and this may be associated with increased sweat gland anaerobic metabolism; c) physical maturation may potentiate the increase in prolactin but not the increase in aldosterone during exercise in the heat.</p> / Doctor of Philosophy (PhD)

<p>Cell-cell adhesion is an important and an initiating event in thrombosis, inflammation and metastasis. Recent data suggest that these adhesion events are mediated by cell adhesion receptors and/or ligands present on blood cells, the endothelium and the basement membrane underlying the endothelium. This thesis hypothesizes that endogenous lipoxygenase metabolites regulate blood cell-vessel wall interactions. The following data, as presented in this thesis, support this hypothesis. First, the intracellular level of 13-HODE in endothelial cells, inversely correlated with platelet, tumor cell and leukocyte adhesion. Second, intracellular 13-HODE levels and/or 13-HODE:HETE ratios in blood cells and in tumor cells also inversely correlated with cell adhesivity. Third, the level of 13-HODE in basement membranes and in artificial grafts, inversely correlated with platelet adhesion. Fourth, 13-HODE co-localized with the vitronectin receptor (VnR) in resting endothelial cells. Following stimulation, 13-HODE and the VnR dissociated, and the VnR relocated to the periphery surface of the endothelial cell. Fifth, the increased peripheral expression of the VnR on stimulated endothelial cells mediated platelet adhesion. Finally modulation of the lipoxygenase pathway markedly influenced experimentally-induced thrombosis. In summary, there is an abundance of data presented in this thesis which suggest that monohydroxy lipoxygenase metabolites regulate blood cell-vessel wall adhesion. This regulation appears to be a result of lipoxygenase metabolites influencing adhesion receptor/ligand recognition. Therefore, it is a reasonable expectation that agents which modulate the lipoxygenase pathway may also be useful in the prevention and treatment of thrombosis, inflammation, and metastasis.</p> / Doctor of Philosophy (PhD)

<p>A phosphotyrosyl-protein phosphatase, designated pTPIII, was purified to near homogeneity from soluble extracts prepared from 11-day old chicken embryo bodies. SDS-PAGE analysis suggested that this pTP activity correlated with the presence of a 58 kd protein band in affinity purified enzyme preparations. The purified protein exhibited biochemical characteristics in common with a phosphotyrosyl-protein phosphatase purified from human placental protein extracts, pTP1B (Tonks et al., 1988a; Pallen et al., 1991), and thus may represent the chicken homolog of this protein. Two partial cDNA clones encoding phosphotyrosyl-protein phosphatases were also isolated. Both code for members of the transmembrane class of tyrosine specific protein phosphatases, and thus probably bear no relation to the pTPIII and pTPI activities purified from soluble protein extracts. One cDNA appeared to encode the COOH-terminus of the chicken homolog of LAR, while the second seemed to code for the chicken homolog of PTP-zeta. Two cDNAs encoding PTP-zeta were isolated, however the evidence presented suggested that both clones were likely derived from incompletely processed nuclear RNAs.</p> / Doctor of Philosophy (PhD)

<p>Rheumatoid arthritis is a chronic, systemic inflammatory disease. It can be treated with the gold(I) based drug, Myochrysine. Much research has been done in the past, in an attempt to elucidate the mechanism of action of this drug. To date, the mechanism remains a mystery. It is known however that Myochrysine inhibits the action of inflammatory cells. Myochrysine has been shown to inhibit phagocytosis, chemotaxis and the respiratory burst in mononuclear phagocytes and polymorphonuclear cells; T and B cell proliferation; interleukin 2 secretion from T cells; interleukin 2 receptor expression on T cells; as well as thrombin stimulated platelet aggregation and release. All of these inflammatory cell functions are mediated by a biochemical pathway involving receptor-G-protein mediated phospholipase C activation which results in Ca²⁺ mobilization and protein kinase C activation. It is proposed that Myochrysine acts to relieve rheumatoid arthritis by interfering with some point(s) in this biochemical pathway in inflammatory cells. Through the use of the platelet as a model system, it has been shown that Myochrysine inhibits the collagen, ADP, and U46619 induced platelet aggregation and serotonin release; that Myochrysine inhibits collagen induced myosin light chain and pleckstrin phosphorylation; that the inhibitory action is fast and reversible; that the inhibitory effect is not dependent on the presence of albumin; that the inhibitory effect does not involve cyclic nucleotide increases and that Myochrysine does not inhibit TPA induced pleckstrin phosphorylation or A23187 induced myosin light chain phosphorylation. More importantly, it is shown that Myochrysine inhibits platelet activation in a manner very similar to inhibition of platelet activation by other sulfhydryl reacting agents, including cell impermeant compounds. The data presented are consistent with the action of Myochrysine being at a membrane surface sulfhydryl group, most probably at a common structurally important sulfhydryl group within platelet receptors involved in the activation of the protein kinase C/Ca²⁺ mobilization pathway. Data are also presented which indicate that both the gold(I) moiety and the thiomalate ligand of Myochrysine can inhibit platelet function.</p> / Doctor of Philosophy (PhD)

<p>In platelets, the activators of guanylyl cyclase used were sodium nitroprusside (SNP) and SIN-1 (the active metabolite of molsidomine). PGE$\sb1,$ a functional analogue of prostacyclin (PGI$\sb2),$ and adenosine were the activators of platelet adenylyl cyclase studied. Changes in cyclic ($\sp3$H) nucleotides were measured in platelets prelabelled with ($\sp3$H) guanine and ($\sp3$H) adenine. Incubation of labelled platelets with SNP or SIN-1 caused inhibitions of platelet function which were associated with large dose-dependent increases in ($\sp3$H) cGMP and 1.4 to 3.0-fold increases in ($\sp3$H) cAMP. However, addition of either SNP or SIN-1 with PGE$\sb1$ or adenosine, at concentrations of the latter that had little effect alone, caused much larger increases in ($\sp3$H) cAMP and greatly enhanced the inhibition of platelet function. Experiments with an adenylyl cyclase inhibitor, 2$\sp\prime,$5$\sp\prime$-dideoxyadenosine (DDA), suggested that these synergistic interactions depend on an enhanced accumulation of cAMP. Hemoglobin inhibited all nitrovasodilator-mediated effects. Cilostamide, a selective inhibitor of platelet cGMP-inhibited cAMP phosphodiesterase (cGI-PDE), had effects identical to those of SNP, suggesting that the actions of the latter depend on inhibition of the same enzyme. Also, the results obtained with M&B 22,948, a cGMP-selective PDE inhibitor, strongly suggest that the increases in platelet ($\sp3$H) cAMP caused by nitrovasodilators, in the presence or absence of activators of adenylyl cyclase, are mediated by the inhibition of cAMP breakdown of cGMP. To determine if a similar synergism is seen in vascular smooth muscle (VSM), the interactions between isoproterenol and either nitrovasodilators or cAMP-PDE inhibitors were studied. A fast and sensitive prelabelling method was established for the measurement of cyclic nucleotides in cultured vascular smooth muscle cells (VSMC) from rat aorta. Using this method, agonist-induced changes in the cyclic nucleotide levels of two different aortic VSMC lines were studied. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)