The present work has endeavored to survey the occurence of adenosine deaminase as well as its multiple forms in normal and some cancerous human tissues. A recent report by Akedo, Nishihara, Shinkai and Komatsu concerning the appearance of the C form adenosine deaminase in cancerous tissues is investigated. The thesis reports, however, the occurence of both A and C forms of adenosine deaminase in almost all normal and cancerous tissues investigated. An increase in C form adenosine deaminase does seem to occur in cancerous tissues, and a ratio method is developed to monitor such an increase in the C form enzyme.Fundamental catalytic and physical parameters are used to characterize the A and C forms of adenosine deaminase in several normal human tissues. Little difference is noted between the two forms of the enzyme. Only substrate specificity for cordycepin is of significant value in differentiating between the A and C forms of the enzyme.Ball State UniversityMuncie, IN 47306

The molecular form distribution of adenosine deaminase has been characterized in nine rat tissues. This study has also investigated the effects of 70% partial hepatectomy in liver, spleen, and intestinal tissues. In all tissues studied, the molecular weight of the enzyme was found to be 35,000 daltons, as determined by gel filtration chromatography. Two prominent isozymes of this molecular weight were identified by means of thin-layer isoelectric focusing. Isozymes with pI's of 4.95 and 4.80 were present in tissues of liver, spleen, and intestine of normal and hepatectomised rats. Throughout the liver regeneration period, activity levels of adenosine deaminase were elevated in all tissues examined.Ball State UniversityMuncie, IN 47306

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

Recent studies demonstrate that there are several G-protein coupled receptors (GPCRs) that dimerize with other GPCRs and form heterodimers. Adenosine A2A-Dopamine D2 receptor interaction is one of the examples for GPCR heterodimerization. Both receptors bear critical roles in physiological processes. Adenosine A2A receptor has functions in neurotransmission, cardiovascular system and immune response. On the other hand, dopamine receptors are the key point of dopaminergic system, which controls the regulation of memory, attention, food intake, endocrine regulation, psychomotor activity and positive reinforcement. Deregulation in dopamine signaling could cause neurological disorders such as Parkinson&rsquo / s disease and schizophrenia. Dopamine D2R and adenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity. The purpose of this study is to optimize Fluorescence Resonance Energy Transfer (FRET) method to detect dimerization of D2R and A2AR by tagging them with EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) in live N2a cell line using laser scanning confocal microscope. Establishing this model will pave the ways for understanding mechanisms of interaction between dopamine and adenosine signaling, thereby, contributing to the understanding molecular mechanisms of some neurophysiological events and disorders. Moreover, the fluorescence based live cell model will be used to detect effects of potential anti-psychotic drugs on the interaction of these two receptors. Indeed, follow-up studies are necessary to extend the limits of this project. Further imaging analyses and drug-receptor interaction studies can be readily applied to extract more information on dopamine-adenosine signaling by using the system developed with this thesis study.

Open Access Article

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 122-137). Also available on the Internet.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 129-143).

Identification of differential expressions of proteins in proteomic profiles of biological samples shows great potential as a valuable technique for the early diagnosis of various diseases. An important challenge in modern protein profiling approaches is to reduce the complexity of the samples by limiting the number of proteins that need to be evaluated for distinction in the expression between normal and deceased cells. In this research, an affinity based approach for the enrichment of nucleotide and nucleoside binding proteins from a complex cell proteome has been developed. To achieve this goal, new N6-biotinylated-8-azido-adenosine probes (AdoRs) have been designed and synthesized to photolabel the nucleotide and nucleoside binding proteins. These probes contain a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Further, a mass spectrometric protein profiling approach is employed to quantitatively identify small variations in expression of nucleoside and nucleotide binding proteins in samples of interest. Mouse neuroblastoma N18TG2 cell proteome has been used as a model system for the development of the LC-MS/MS based proteomic analysis of these affinity enriched protein fractions. Upon enrichment, the photolabeled proteome exhibited an approximately four-fold abundance of nucleoside and nucleotide binding proteins over nonlabeled proteome. The approach was extended to compare the proteomic profiles of nucleotide and nucleoside binding proteins in cancerous (Hey) and non-cancerous (T-80) human ovarian cell proteome. Certain proteins that were not detected in cell lysate were also identified in labeled proteome, thereby demonstrating the strength of our approach in enriching low abundant proteins. To substantiate the qualitative analysis, we have employed the Stable Isotope Labeling in Amino Acid Cell Culture (SILAC) for the quantitative study of the protein expression in cancerous and non-cancerous human ovarian cells. A modest panel of proteins with differential expressions in these cell lines was identified, a few of which have been correlated to various forms of cancer. Vimentin, stress induced phosphoprotein-1, and heat shock protein 90 that were identified to have altered expressions in these cell lines are among some of the proteins associated with ovarian cancer.

The cystic fibrosis transmembrane conductance regulator (CFTR) was identified to be involved in acidosis-induced ATP release from skeletal myocytes in vitro and from contracting muscle in vivo. My PhD studies aimed to investigate the underlying mechanism and identify the pathway for ATP release in acidosis-induced CFTR-regulated ATP release. Lactic acid (10 mM) decreased the intracellular pH of L6 skeletal myocytes to 6.87 ± 0.12 after 3 hours, and the lowered pH resulted in the elevation of ATP release from skeletal myocytes. The acidosis-induced ATP release was totally abolished by GlyH-101 (40 μM), an open-channel CFTR blocker, suggesting that CFTR was involved. The cAMP/PKA signaling pathway was involved in the CFTR-regulated ATP release from skeletal myocytes: 1). Forskolin increased the extracellular ATP and the phosphorylation of CFTR; IBMX, a phosphodiesterase inhibitor, further enhanced the forskolin-induced extracellular ATP and phosphorylation of CFTR; 2). Inhibition of PKA by its selective inhibitor KT-5720 abolished the acidosis-induced ATP release and the forskolin-induced phosphorylation of CFTR. In addition, the inhibition of Na+/H+ exchanger (NHE) by amiloride, or inhibition of Na+/Ca2+ exchanger (NCX) by its specific inhibitors SN-6 and KB-R7943 abolished the lactic-acid-induced ATP release from skeletal myocytes, indicating that NHE and NCX might be involved. Previous studies demonstrated that Connexin hemichannels and Pannexin channels were able to conduct ATP in response to stimuli. This study found that connexin 43 (Cx43) was strongly expressed on skeletal myocytes, while Pannexin 1 (Panx1) showed a strong expression in gastrocnemius muscle. Investigation of the role that Cx43 may play in acidosis-induced cAMP/PKA-activated CFTR-regulated ATP release from myocytes showed that: 1). Cx43 was immunoprecipitated with CFTR suggesting a physical interaction; 2). The opening of Cx hemichannels was increased by lactic acid and this lactic-acid-induced opening was inhibited by CFTRinh-172, suggesting the mediation of CFTR; 3). Inhibition of Cxs and Panxs with carbenoxolone abolished the acidosis-induced ATP release; moreover, specific silencing of the Cx43 gene using siRNA decreased both basal and acidosis-induced ATP release, suggesting that Cx43 was involved; 4). Overexpression of CFTR alone did not elevate the acidosis-induced ATP release, while overexpression of Cx43 alone doubled the acidosis-induced ATP, and co-overexpression of CFTR and Cx43 further elevated the acidosis-induced ATP release, supporting the concept that Cx43 functionally interacted with CFTR to induce the acidosis-induced ATP release. Panx1 was studied in native skeletal muscle, and found to be coimmunoprecipitated with CFTR. Inhibition of Panxs with gadolinium or probenecid abolished the muscle-contraction-induced ATP release, while inhibition with carbenoxolone or quinine reduced it to less than 10% of control, suggesting that Panx1 may be involved in the acidosis-induced ATP release during muscle contraction. All the in vitro and in vivo studies suggested that Cxs and Panx were involved in the acidosis-induced CFTR-regulated ATP release from skeletal myocytes and skeletal muscle. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy