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The role of cyclic AMP and a differentiation-indueing factor (DIF) in the differentiation of stalk cells was investigated in the cellular slime mold Dictyostelium discoideum. In this organism, starvation triggers the aggregation of amoebae into multicellular masses within which a simple, well-regulated pattern of partially differentiated cells is formed and which ultimately form fruiting bodies comprised of spore and stalk cells. In a monolayer system at low cell densities, stalk cell formation is dependent on the presence of both cyclic AMP and DIF. Both factors act within a short time of each other, induction by cyclic AMP preceding induction by DIF, beginning between 8 to 10 hours of incubation in monolayers, and progressively committing an increasing proportion of the cells in monolayer to form stalk cells. The relative effectiveness of analogues of cyclic AMP to induce stalk cell formation in monolayers indicates that the well-characterized cell surface cyclic AMP receptor most probably mediates the action of cyclic AMP. Although this receptor appears early during aggregation, it does not become activated until later during development in vivo, probably because the cyclic AMP concentrations within developing cell masses must build up to levels higher than those in aggregation streams. The finding that caffeine inhibits stalk cell formation in low density monolayers and that the permeable analogue 8-Bromo-cyclic AMP can partially reverse this inhibition suggests that activation of this receptor leads to an increase in internal cyclic AMP levels as one of the steps in stalk cell differentiation. The finding that the expression in low density monolayers of AP IV, a cell-type non-specific isozyme of acid phosphatase, was cyclic AMP-dependent is consistent with the view that cyclic AMP induces non-specific postaggregative gene expression during development in vivo. The findings that the expression of pre-stalk arid stalk cell specific antigens and of the pre-stalk cell specific isozyme AP II was DIF-dependent provide good evidence for the idea that both pre-stalk and stalk cell formation are induced by DIF. The fact that isolated pre-stalk cells require DIF for stalk cell formation in low density monolayers further supports this idea. Whereas cells independent of DIF for stalk cell formation in monolayers appear immediately after cyclic AMP-independent cells during differentiation in low density monolayers, DIF-independent cells appear considerably later during development in vivo. This evidence and the fact that developing cell masses contain elevated levels of DIF lead to the postulate that the factor(s) which triggers the formation of fruiting bodies also controls the pre-stalk to stalk cell conversion. / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

Improved sequencing technologies have created an explosion of sequence information that is analyzed and proteins are annotated automatically. Annotations are made based on similarity scores to previously annotated sequences, so one misannotation is propagated throughout databases and the number of misannotated proteins grows with the number of sequenced genomes. A systematic approach to correctly identify the function of proteins in the amidohydrolase superfamily is described in this work using Clusters of Orthologous Groups of proteins as defined by NCBI. The focus of this work is COG1816, which contains proteins annotated, often incorrectly, as adenosine deaminase enzymes. Sequence similarity networks were used to evaluate the relationship between proteins. Proteins previously annotated as adenosine deaminases: Pa0148 (Pseudomonas aeruginosa PAO1), AAur_1117 (Arthrobacter aurescens TC1), Sgx9403e and Sgx9403g, were purified and their substrate profiles revealed that adenine and not adenosine was a substrate for these enzymes. All of these proteins will deaminate adenine with values of kcat/Km that exceed 105 M-1s-1. A small group of enzymes similar to Pa0148 was discovered to catalyze the hydrolysis of N-6-substituted adenine derivatives, several of which are cytokinins, a common type of plant hormone. Patl2390, from Pseudoalteromonas atlantica T6c, was shown to hydrolytically deaminate N-6-isopentenyladenine to hypoxanthine and isopentenylamine with a kcat/Km of 1.2 x 107 M^-1 s^-1. This enzyme does not catalyze the deamination of adenine or adenosine. Two small groups of proteins from COG1816 were found to have 6-aminodeoxyfutalosine as their true substrate. This function is shared with 2 small groups of proteins closely related to guanine and cytosine deaminase from COG0402. The deamination of 6-aminofutalosine is part of the alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminofutalosine is deaminated with a catalytic effeciency of 105 M-1s-1 or greater, Km’s of 0.9 to 6.0 µM and kcat’s of 1.2 to 8.6 s-1. Another group of proteins was shown to deaminate cyclic- 3’, 5’ -adenosine monophosphate (cAMP) to produce cyclic-3’, 5’-inosine monophosphate, but will not deaminate adenosine, adenine or adenosine monophosphate. This protein was cloned from a human pathogen, Leptospira interrogans. Deamination may function in regulating the signaling activities of cAMP.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis advisor: Charles Hoffman / In both mammals and fission yeast, control of cAMP levels is maintained by adenylyl cyclases (ACs), which synthesize cyclic nucleotide, and by cyclic nucleotide phosphodiesterases (PDEs), which are responsible for its degradation. AC activity is regulated by G proteins, which respond to signals from G protein-coupled receptors (GPCRs) that detect extracellular signaling factors such as hormones. cAMP is a second messenger that has several effectors, with protein kinase A (PKA) being a primary target of activation that phosphorylates several downstream targets and results in modulation of pathways such as cell growth and gluconeogenesis. Aberrant cAMP regulation has been linked to several human disease states, such as McCune-Albright Syndrome, which is the result of elevated cAMP levels. Whereas the targeting of PDEs with drugs and selective inhibitors has been very successful, the AC-inhibiting compounds identified to date are unfavorable for clinical use. Inhibitors may not necessarily bind to and inhibit a given AC directly but instead act on a regulatory pathway such as calmodulin signaling. Theoretically, they also may bind to the G protein, interfere with the AC-G protein stimulatory complex, or regulate a factor of AC transcription. Since more than one AC species is expressed in many human cell types, it is difficult to selectively reduce cAMP levels. Therefore, for an AC inhibitor to be favored as a candidate for drug development, it is likely that the compound should directly bind to and inhibit the AC. This thesis describes my studies on a scaffold of 41 structurally related BCAC compounds, called the BCAC51 scaffold, that was identified in a high-throughput screen (HTS) with Schizosaccharomyces pombe strains transformed with GNAS and either mammalian AC4 or AC7. I carried out a series of experiments to examine whether the compounds bind to and inhibit mammalian ACs directly. The most active compounds were further characterized for potency and specificity against a panel of ACs. Several compounds significantly reduced cAMP production, but it could not be determined if the compounds directly or indirectly altered AC activity. I also cloned and constructed strains expressing the human wild-type AC5 gene and the AC5 R418W mutant, which has shown an increased sensitivity to GNAS. cAMP assays on these strains using various BCAC compounds showed that while most compounds had similar effects on both forms of AC5, BCAC62 was significantly more effective on the wild-type enzyme than on the mutant AC5, although the reason for this is unclear. To test whether the compounds could reduce AC activity in the absence of GNAS (basal activity), a flow cytometry study was carried out using a PKA-repressed GFP reporter. Results suggested that BCAC compounds do reduce basal-AC activity and therefore do not act by binding to and inhibiting GNAS, by interfering with the AC-GNAS stimulatory complex, nor by stimulating PDE. Finally, I developed a molecular genetic screen for mutant alleles of an AC gene that confer compound-resistance. One cycle of the screen is near completion, and the screen provides a foundation for future examination of compound-resistant AC candidates. The results presented in this thesis serve as a basis for further research into members of the BCAC51 compound series being putative direct inhibitors of mammalian ACs. / Thesis (BS) — Boston College, 2022. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20663号 / 医博第4273号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤渕 航, 教授 木村 剛, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains iv, 60 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19806号 / 人博第777号 / 新制||人||187(附属図書館) / 27||人博||777(吉田南総合図書館) / 32842 / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM