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In the present study, adenosine kinase (AK) was purified from a number of different sources (e.g. bovine liver, Syrian hamster liver, kidney and heart; Chinese hamster ovary cells and human placenta). The enzyme from bovine liver and Syrian hamster liver was purified to apparent homogeneity by a combination of ion-exchange chromatography, affinity chromatography and gel filtration chromatography. The purified enzyme showed a single band of 40 kDa in SDS-polyacrylamide gel electrophoresis, which was similar to that reported from other sources in the literature. A number of biochemical and enzymatic characteristics of AK were investigated. The reliability and reproducibility of the AK assay as established in previous studies was determined. The apparent Km of partially purified AK from Syrian hamster liver for adenosine was determined to be 0.16 uM, which is consistent with earlier reports. A novel result obtained in these studies is that AK activity was found to be completely dependent upon the presence of phosphate or other pentavalent ions. AK from different sources did not show any activity in the Tris-HCl buffer, pH 7.0, but the activity increased dramatically upon the addition of phosphate and it reached a maximum at 2 mM phosphate. There was no inhibition of AK activity when the phosphate concentration was increased up to 100 mM. AK could also be activated by substituting phosphate with either arsenate or vanadate, which have similar chemical structures to phosphate. The temperature inactivation kinetics of AK showed that AK from human fibroblast cells had higher thermal resistance than AK from Chinese hamster ovary cells at 50°C. The presence of phosphate had no effect on the thermal stability of AK. Antibodies to purified AK from Syrian hamster liver were raised in both rabbits and guinea pigs. Antiserum from the rabbit which gave the strongest response was used for further studies. AK was recognized by and reacted specifically with the antiserum at a dilution of up to 1: 16,000. The AK antibody which was covalently bound to Protein A Sepharose beads immunoprecipitated a 40 kDa protein from radio-labelled Chinese hamster ovary or baby hamster kidney cell extracts. In addition, this antibody preparation immunoprecipitated AK activity from Syrian hamster liver extracts. However, immunoblotting showed that all the antisera could not react with AK from Chinese hamster ovary cells and human cells, suggesting a strong species specificity. A partial protein sequence of AK was obtained by microsequencing of a cyanogen bromide fragment of purified AK from Syrian hamster liver. The sequence was Tyr-Val-Asp-Ser-Leu-Phe-Gly-Ala-Glu-Thr-Glu-Ala-Ala-Leu. Degenerate DNA probes for this sequence can now be made and used for either screening of eDNA libraries or carrying out PCR experiments to facilitate the cloning of the AK gene(s). Finally, conditions for selection of revertants from AK⁻ mutants of Chinese hamster ovary cells have been developed. The AK⁺ revertants from AK⁻ mutants can be obtained using adenosine, alanosine, uridine and erythro-9-(2-hydroxyl-3-nonyl) adenine in the growth medium. Three revertants have been isolated. These revertants regained their AK activities and lost their drug-resistance at same time. This method can also be used in the future to clone the AK gene by transfecting AK⁻ mutants with foreign DNA. / Thesis / Master of Science (MSc)

Le développement tumoral nécessite une dérégulation des contrôles normaux de la prolifération et de la différenciation cellulaires. Il est bien connu que la voie de signalisation ERK/MAP kinase est impliquée dans ces processus de régulation cellulaire. De plus, un rôle essentiel dans la transformation cellulaire est attribué à MEK1 qui est un élément central de cette voie. Une meilleure compréhension de l'implication de MEK1 dans les voies de transduction devrait donc nous permettre de mieux comprendre le developpement cellulaire et la transformation morphologique. Mes travaux de recherche tentent d'élucider les mécanismes de régulation des protéines kinases MEK1 et MEK2 dans le but de mieux comprendre leur divergence fonctionnelle et leur implication dans les différentes réponses cellulaires. Ainsi, l'étude de la voie ERK/MAPK chez les fibroblastes embryonnaires mutants pour le gène Mek1 indique que la transduction du signal amorcée par un neuropeptide, la bombésine, passe spécifiquement par MEK1 et serait indépendant de MEK2. La région C-terminale de MEK1 semble médier la spécificité de la réponse à la bombésine. Le domaine MSS, qui est une insertion d'une séquence riche en proline unique à MEK1 et MEK2 pourrait être la clef de cette réponse spécifique. En outre, nos travaux de délétion et d'interactions protéiques suggèrent qu'une variation de la conformation de la région C-terminale de MEK1 pourrait avoir lieu entre l'état inactif et l'état actif de ces MAPKKs. En absence d'activation, la région en caboxy du domaine kinase semble interagir avec la boucle d'activation se trouvant au cœur du domaine kinase. Cette interaction intramoléculaire serait dépendante de l'état de phosphorylation de MEK1. Par contre, la région en carboxy ne semble pas être un domaine d'autoinhibition ou un pseudo substrat puisque sa délétion ne met pas la protéine dans un état constitutivement actif. Ainsi, il est possible que cette région soit essentielle du point de vue structural pour permettre, en fonction de son activité, la régulation des interactions de MEK1 (ou MEK2) avec ses activateurs, substrats ou protéines d'échafaudage.

A compelling mystery in the study of exercise is mechanisms of skeletal muscle fatigue. Broadly described, muscle fatigue is the uncomfortable sensation that particular muscle groups are shutting down and muscle force production cannot continue. More specifically, muscle fatigue is defined as an activity-induced inability to continue to produce a desired level of force. Several groups suggest that a major cause of force loss during fatigue is reductions in the rates of sarcoplasmic reticulum (SR) calcium (Ca2+) release and uptake. These changes result in diminished contractile machinery activation, reduced force production and slowed relaxation. During exercise, adenosine 5'-triphosphate (ATP) is the energy currency that is used to support force production. As a result of ATP hydrolysis and re-synthesis, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) levels rise. AMP kinase (AMPK) is an enzyme that becomes activated as a result of increased AMP levels. It is thought to function as a metabolic "master switch" within the muscle and plays a major role in carbohydrate and fat metabolism. Once AMPK is activated it regulates several ATP consuming and producing pathways. The overall objective of this project was to determine if increased metabolism during exercise contributes to SR Ca2+ dysfunction during fatigue. If this is true, artificial activation of AMPK via 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) should induce changes in SR function that are qualitatively similar to those caused by fatigue. In study 1, mice were injected with 0.85 mg/kg AICAR (or saline solution) and both gastrocnemius muscles were removed one hour later. In study 2, EDL muscles were placed in a muscle bath and incubated in AICAR (4mM) or stimulated to fatigue. Glycogen, glucose-6-phosphate (G-6-P), ATP, ADP, and phosphocreatine (PCr) were examined in all groups of muscles. Alterations in SR calcium uptake and release rates due to the presence of AICAR were also studied as a likely cause of muscle fatigue. AICAR treatment in vivo did not alter muscle glycogen, glucose, ATP, ADP or PCr concentrations. However, G-6-P levels were increased by 137%. This was accompanied by a 36% reduction in the SR Ca2+ uptake rate and a 42% reduction in Ca2+-stimulated Ca2+ ATPase activity as well as 13-15% reductions in the rates of Ca2+ release. These changes were not associated with SR Ca2+ pump content. Administration of AICAR in vitro also increased G-6-P content (200%) without altering the concentrations of glycogen, glucose, G-6-P, ATP, ADP or PCr. AICAR decreased SR Ca2+ uptake rate by 28% and the rate of Ca2+ release by 16%. For comparison, fatiguing stimulation reduced the rates of SR Ca2+ uptake and release by 31 and 41%, respectively. Taken together, these results indicate that when administered to skeletal muscle both in vivo and in vitro, AICAR evokes metabolic stress. More importantly, activation of AMPK alters skeletal muscle SR function to an extent that is qualitatively similar to that caused by fatiguing activity. At present, it is not clear how AMPK activation causes changes in SR function. However, the present finding is consistent with the notion that metabolic stress caused by exercise, affects SR function. This, in turn, leads to force loss but reduces energy demand and protects the cell from ATP depletion during maximal contractile activity. / Master of Science

Using the genome database of the phytopathogenic fungus, Alternaria brassicicola, we identified a gene with high homology to the cell wall integrity-associated mitogen-activated protein (MAP) kinase, Slt2 in the yeast, Saccharomyces cerevisiae. This MAP kinase consists of a predicted 1,251-bp open reading frame, and encodes a 416-amino-acid protein weighing 47501 Da. This homolog was designated AbSlt2 (A. brassicicola Slt2) and gene disruption knockout (KO) mutants were generated in an A. brassicicola wild type background. Several altered phenotypes were found in the mutants compared to the wild type. During growth in various liquid and solid media, the abslt2 mutants displayed slightly aberrant hyphal growth and were unable to develop at the same rate as wild type. Furthermore, scanning electron microscopy (SEM) analysis revealed the abslt2 mutants showed decreased penetration ability, underdeveloped appresoria, and altered morphology on the leaf surface of the host plant, Brassica oleracea (cabbage) when compared to wild type. Abslt2 mutant hyphae exhibited slower growth in planta ultimately resulting in highly reduced virulence. Complementation of the disruption mutant with the wild type gene fully restored pathogenicity. Therefore, AbSlt2 is a new pathogenicity and developmental factor in A. brassicicola. / Master of Science

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 118-130.