Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC.

N-Ethylcarboxamidoadenosine (12) was synthesised from adenosine (1) and the 6-chloro-2’,3’-O-isopropylidene-AT-ethylcarboxamidoadenosine (25) was synthesised from inosine (19). Employing molecular modelling techniques and the results from previous structure activity relationships it was possible to design and synthesise a N6-substituted N-ethylcarboxamidoadenosines which possessed an oxygen in the N6-substituent either in the form of an epoxide (which was obtained by cpoxidising an alkene with m-CPBA or dimethyldioxirane) or in the form of a cyclic ether as was the case for N6-((tetrahydro-2H--pyran--2-yl)methyl-N-ethylcarboxamidoadenosine (78). These compounds were tested for their biological activity at the A1 adenosine receptor by their ability to inhibit cAMP accumulation in DDT, MF2 cells. The EC50 values obtained indicated that the N6-(norborn-5-en-2-yl)-N-ethylcarboxamidoadenosines were the most potent. Of theseN6-(S-endo-norbrn-5-en-2-yI)-N-ethylcarboxaniidoadenosine (56) was the most potent (0.2 nM). N6-(exo-norborn-5-en-2-yl)-2-iodo-N-ethylcarboxamidoadenosine (79) was synthesised from guanosine (22) and was also evaluated for its potency at the A, receptor (24.8 ± 1.5 nM). At present 79 is being evaluated for its selectivity for the A1 receptor compared to the other three receptor subtypes (A2a, A2b, A3). A series of N6-(benzyl)-N-ethylcarboxamidoadenosines were synthesised with substitutions at the 4-position of the phenyl ring. Another series of compounds were synthesised which replaced the methylene spacer between the N6H and the N6-aromatic or lipophilic substituent The replacement groups -were carbonyl and trans-2- cyclopropyl moieties. The N6-acyl compounds were obtained by reacting 2’,3’-O- di(tert-butyldimethylsilyl)-AT-ethylcarboxamidoadenosinc (59) with the appropriate acid chloride and then deprotecting with lelrabutylammonium fluoride in tetrahydrofuran. The compound N6-(4-(1,2-dihydroxy)ethyl)benzyl-N- ethylcarboxamidoadenosine (125) was synthesised by the reaction of 4-(1,2-0- isopropylidene-ethyl)benzyl aminc (123) with 6-chloro-2,3-0-isopropylidene-N- ethylcarboxamidoadenosine (25). Compound 123 was synthesised from an epoxidation of vinylbenzyl phthalimide (118) followed by an acidic ring opening to yield the diol which was isopropylidenated to yield 4-(l,2-O-isopropylidene- elhyl)benzyl phlhalimide (122), It was hoped that the presence of the diol functionality in 125 would increase water solubility whilst maintaining potency at the A3 receptor.

Adenosine (Ado) salvage is essential in plant development. The lack of Ado kinase activity (ADK) in Arabidopsis thaliana adk1 adk2 double mutants results in embryonic lethality; reduction of ADK expression causes a pleiotropic phenotype due to the accumulation of Ado inhibiting transmethylation activities. The phenotype of ADK mutants shows that this enzyme plays a critical role in Ado salvage but the functional significance of the other putative Ado recycling enzymes Ado deaminase (ADA) and Ado nucleosidase (ADN) in Arabidopsis thaliana have yet to be elucidated. ADA catalyzes the irreversible deamination of Ado to inosine. The locus At4g04880 (AtADA) of A. thaliana is annotated as encoding a putative ADA, based on its amino acid sequence similarity and the presence of important, conserved catalytic residues. However, indirect and direct spectrophotometric activity assays of the recombinant enzyme demonstrated that the gene product of this locus does not possess ADA activity; complementation experiments to test for the functionality of the AtADA product in A. thaliana and E. coli confirmed its lack of ADA activity. Instead, phylogenetic analysis revealed that AtADA belongs to the group of ADA-like (ADAL) proteins, a group closely related to ADAs that to date have not been shown to have ADA activity. AtADA is no exception as it also lacks ADA activity based on the in vivo and in vitro experiments outlined in this thesis. Thus, the locus At4g04880 should be re-annotated as ADAL. The question of the function of AtADAL cannot be answered as of yet; in general, the knockout of ADA gene product demonstrated that At4g04880 is not essential for Arabidopsis growth. Since no further ADA-related genes exist in the genome of Arabidopsis it is concluded that ADA activity is not present in this plant. ADN catalyzes the conversion of purine and pyrimidine ribosides to their corresponding bases; although it prefers Ado as a substrate it also acts on cytokinins. The activity of this enzyme has been described in several plant species but no corresponding genes have been identified to date. The genome of Arabidopsis was screened for ADN genes using an inosine-uridine nucleoside hydrolase sequence from the protozoa Crithidia fasciculata. Two genes, annotated as ADN1 and ADN2 were identified and their gene products were studied using a spectrophotometric assay. The substrate spectrum of ADN2 includes both purine and pyrimidine nucleosides but it prefers to utilize uridine. Thus, ADN2 is proposed to be involved in the purine and pyrimidine salvage in Arabidopsis but predominantly in uridine recycling. Recombinant ADN1 did not show activity on any of the tested substrates. Even though the in vivo role of both ADNs is still uncertain, due to their lack or low activity on Ado there may yet be the ADN gene in the Arabidopsis genome which likely acts on both adenosine and cytokinin ribosides.

Adenosine (Ado) salvage is essential in plant development. The lack of Ado kinase activity (ADK) in Arabidopsis thaliana adk1 adk2 double mutants results in embryonic lethality; reduction of ADK expression causes a pleiotropic phenotype due to the accumulation of Ado inhibiting transmethylation activities. The phenotype of ADK mutants shows that this enzyme plays a critical role in Ado salvage but the functional significance of the other putative Ado recycling enzymes Ado deaminase (ADA) and Ado nucleosidase (ADN) in Arabidopsis thaliana have yet to be elucidated. ADA catalyzes the irreversible deamination of Ado to inosine. The locus At4g04880 (AtADA) of A. thaliana is annotated as encoding a putative ADA, based on its amino acid sequence similarity and the presence of important, conserved catalytic residues. However, indirect and direct spectrophotometric activity assays of the recombinant enzyme demonstrated that the gene product of this locus does not possess ADA activity; complementation experiments to test for the functionality of the AtADA product in A. thaliana and E. coli confirmed its lack of ADA activity. Instead, phylogenetic analysis revealed that AtADA belongs to the group of ADA-like (ADAL) proteins, a group closely related to ADAs that to date have not been shown to have ADA activity. AtADA is no exception as it also lacks ADA activity based on the in vivo and in vitro experiments outlined in this thesis. Thus, the locus At4g04880 should be re-annotated as ADAL. The question of the function of AtADAL cannot be answered as of yet; in general, the knockout of ADA gene product demonstrated that At4g04880 is not essential for Arabidopsis growth. Since no further ADA-related genes exist in the genome of Arabidopsis it is concluded that ADA activity is not present in this plant. ADN catalyzes the conversion of purine and pyrimidine ribosides to their corresponding bases; although it prefers Ado as a substrate it also acts on cytokinins. The activity of this enzyme has been described in several plant species but no corresponding genes have been identified to date. The genome of Arabidopsis was screened for ADN genes using an inosine-uridine nucleoside hydrolase sequence from the protozoa Crithidia fasciculata. Two genes, annotated as ADN1 and ADN2 were identified and their gene products were studied using a spectrophotometric assay. The substrate spectrum of ADN2 includes both purine and pyrimidine nucleosides but it prefers to utilize uridine. Thus, ADN2 is proposed to be involved in the purine and pyrimidine salvage in Arabidopsis but predominantly in uridine recycling. Recombinant ADN1 did not show activity on any of the tested substrates. Even though the in vivo role of both ADNs is still uncertain, due to their lack or low activity on Ado there may yet be the ADN gene in the Arabidopsis genome which likely acts on both adenosine and cytokinin ribosides.

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 87-93). Also available for download via the World Wide Web; free to University of Oregon users.

VCP/p97 works as a segregase to extract the ubiquitylated proteins from protein complexes, lipid membranes and chromosomes, thereby promoting their degradation or recycling. VCP/p97 plays essential roles in ubiquitin-dependent proteasome degradation, ERAD, autophagy, endocytosis, reassembly of ER, Golgi and nuclear envelop, and cell cycle regulation. In ubiquitin dependent proteasome degradation pathway, VCP/p97, as a special ubiquitin binding-shuttle factor, is required for the successful cell cycle progression in regulating IκB, CDT-1, Aurora B, and CDC25A. Here, we studied the role of VCP/p97 in G1 to S phase transition in MCF-7 human breast cancer cells. We found that VCP/p97 knockdown or inhibition by DBeQ, a potent VCP/p97 inhibitor, decreased cell proliferating rates and reduced S phase cell percentages in asynchronized MCF-7 cells.VCP/p97 inhibition by DBeQ also arrested cells at G1 phase in synchronized MCF-7 cells. These data suggest that VCP/p97 is required for G0/G1 to S phase transition in MCF-7 cells. In addition, in either asynchronized or synchronized MCF-7 cells, VCP/p97 knockdown or DBeQ treatment resulted in the accumulation of p21 and p27, two CDK inhibitors. Moreover, p27, not p21, knockdown in MCF-7 cells rescued the defects of S phase entry caused by VCP/p97 knockdown or DBeQ treatment. Taken together, our results suggest that VCP/p97 regulates the timely degradation of p27 to promote G1 to S phase transition in MCF-7 cells. / published_or_final_version / Physiology / Master / Master of Philosophy

The tonoplast H$ sp+$-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides (57 and 67 kDa). Kinetic analysis of ($ alpha$-$ sp{32}$P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ($ sp{14}$C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H$ sp+$-ATPase is a multimer composed of at least three polypeptides. / Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H$ sp+$-ATPases of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F$ sb0$F$ sb1$ and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

Adenosine is a nucleoside with varied pharmacological effects but its actions in man are sparsely documented. Cardiac and respiratory effects of adenosine in man were examined. Adenosine was found to increase coronary flow in patients without significant coronary atheroma. Adenosine restored sinus rhythm in some patients with supraventricular tachycardia. In those in whom sinus rhythm was not restored the underlying rhythm was atrial flutter. In patients with complete heart block adenosine decreased ventricular rate in a dose-related fashion. In subjects in sinus rhythm adenosine produced a transient dose-related bradycardia which was followed by a more sustained increase in sinus rate. These latter effects were compared but were found not to differ in young and elderly subjects. A dose-related respiratory stimulant effect of adenosine, which had not previously been widely appreciated, was observed. The possible relevance of this observation to the ventilatory response to hypoxia is discussed. Adenosine-induced respiratory stimulation was found not to differ in young and elderly subjects. Dipyridamole, an inhibitor of adenosine transport, potentiated adenosine-induced respiratory stimulation and bradycardia but not the subsequent tachycardia. Aminophylline, a competitive adenosine antagonist at cell-surface receptors, abolished adenosine-induced bradycardia but did not alter the tachycardia or respiratory stimulation. Adenosine-induced bradycardia in man may be explained by an action of adenosine on cell-surface receptors at one site, whereas such an explanation does not accord with the observations on tachycardia and respiratory stimulation. Adenosine administered proximal to the carotid circulation in man stimulated respiration, but infusion distal to those vessels had no such effect. These observations are consistent with the hypothesis that adenosine stimulates respiration in man by an action on the carotid body. Possible physiological, pathophysiological and therapeutic implications of these observations are discussed.

Two molecular forms of adenosine deaminase have been isolated from bovine livers. These two forms were found to be interconvertible. Adenosine deaminase extracted from human tissues exhibited similar properties as well. Previous studies have shown the presence of a conversion factor which formed an aggregate with the small form (the C-form) of the enzymes, and the resulting enzyme complex was identified to be the large form (the A-form) of adenosine deaminase. Studies have been made with rat tissues. The C-form of the adenosine deaminase is widely distributed in the various tissues, while the large form of the enzyme is absent in those tissues examined.The outline of this study is summarized below: (1) The isolation of the conversion factor from human liver.(2) The isolation of the adenosine deaminase from the various tissues.(3) The coversion of human adenosine deaminase C-form to the A-form.(4) Quantization of the conversion of the C-form to the A-form.(5) Testing the effect of conversion on nonhuman enzymes.(6) The optimum temperature and time to yield greatest amount of conversion.The results from this study would verify any differences that might exist between the small forms of the enzyme present in different species.

Cytochemical localization of nuclear-envelope nucleos-side triphosphatase was exhibited in the nuclear membrane and nuclear pores of the Necturus, maculosus oocyte. This enzyme is thought to promote nucleocytoplasmic transport of mRNA through the nuclear pores. Various tissue preparations were performed to assure optimum results of reaction product formation and preservation of tissue ultrastructure.Incubation of frozen oocyte sections in a modified Wachstein-Meisel medium resulted in positive staining of the nuclear membrane and the nucleoli as evidenced in light and electron micrographs. Whole oocytes were incubated in the Wachstein-Meisel medium and then embedded in Epon for electron microscopy. The whole oocytes contained reaction product associated with microvilli and plasma membranes. Exposure of manually isolated nuclei to the same experimental medium resulted in lead deposits in the nuclear envelope, the nuclear pores, and randomly dispersed among chromatin granules. Thus, these nuclear structures may play a role in transport of mRNA to the cytoplasm. / Department of Physiology and Health Science