This study was designed to: 1. Examine the diagnostic value of ADA levels in ascitic fluid, 2. Establish the sensitivity and specificity of this test in the diagnosis of tuberculous peritonitis, in a large number of patients, 3. Establish levels of adenosine deaminase activity which give the best discriminatory information in patients with ascites. 4. Determine what conditions may give rise to false positive or false negative results. 5. Finally, the study was designed to assess the relative diagnostic accuracy of previously used biochemical and haematological data, such as ascites total protein and white cell count. The diagnostic value of these tests alone, and combined with ADA activity in a descriminant analysis, was compared with the diagnostic accuracy of adenosine deaminase activity alone.

No description available.

No description available.

Adenosine kinase (AK) is a purine salvage enzyme which catalyses the phosphorylation of the 5'-hydroxyl of adenosine via ATP. AK is a key enzyme which controls the intra and extracellular concentration of adenosine (Ado). Agents which inhibit the activity of AK have been found to attenuate cellular damage, demonstrating therapeutic utility in a variety of disease processes. In order to design inhibitors of AK with increased efficacy, a better understanding of enzyme activity is required. Previously, a number of novel characteristics of mammalian AK had been discovered. It was shown that the activity of AK is influenced by the presence of pentavalent ions (PVD such as inorganic phosphate (Pi), arsenate and vanadate. A detailed study of the influence of Pi on the kinetic parameters of Chinese hamster (CH) and beef liver AK was performed. These studies suggested that the Km (Ado) decreases and the Ki (Ado) increases asymptotically in the presence of increasing concentrations of Pi. Under the same conditions, the Vmax for activity increases hyperbolically. The effect of phosphate is not limited to the mammalian form of AK. Pi, arsenate and vanadate were all found to have similar effects on AK from yeast, spinach and Leishmania donovani AK. PVI as well as the metabolite phosphoenol pyruvate were also found to stimulate the activity of the enzyme ribokinase (RK) from E. coli, which similar to AK, is a member of the PfkB family of carbohydrate kinases. Although AK and RK show little sequence similarity, the residues at the active site and the 3D structues of these two proteins are very similar. Based on sequence alignment of PfkB family members, we have identified a conserved sequence motif, NXXE, which based upon the available structural information appears to be involved in the binding of phosphate. To confirm and understand the role of this motif in Pi binding, the residues at the NXXE site were altered by site-directed mutagenesis and their effect on activity of the recombinant CH AK was examined. Though the residues at the NXXE site do not directly interact with substrate, nor the putative catalytic base, the resulting proteins were found to have greatly altered phosphate requirement, substrate inhibition characteristics and different magnesium requirements. In the AK structure, aspartic acid at position 316 is presumed to act as the catalytic base. This residue was changed to asparagine and glutamic acid by mutagenesis. The resulting proteins were found to be nearly completely devoid of activity, confirming its critical role in AK activity. The amino acid sequence at the extreme N-terminus of AK has been found to exhibit the greatest variability within and among species, though the rest of the protein remains greatly conserved. To delineate the residues that are involved in the structural stability and activity of AK, systematic deletions of the residues from both the N-and C-terminus were performed, and the structure-activity relationships were examined. It was determined that the first 16 residues of CH AK can be removed without affecting activity. Removal of the next 11 residues resulted in sequential decreases in enzyme stability and activity. These 11 residues are involved in the first B-structure of the protein and are required for the stability of the tertiary structure. All residues at the C terminus were required for activity, and involved in a hydrogen-bonding network necessary for the stability of the ATP binding site. These studies provide novel insight to the structure-activity relationship of mammalian AK as well as the PfkB family of enzymes. Our work has identified a site distal to the catalytic site, that is implicated in the PVI binding and catalytic effect. Further studies should be aimed at understanding how binding of PVI at this site influences the catalytic activity of AK. Development of inhibitors which bind to this site and modulate the activity of AK should prove very useful in this regard. / Thesis / Doctor of Philosophy (PhD)

While previous research into the pathogenesis of ischaemic and reperfusion injuries has focussed on the cardiac myocyte, there is increasing evidence for a crucial role for coronary vascular injury in the genesis of the post-ischaemic phenotype [1-3]. Post-ischaemic vascular injury may be manifest as a transient or sustained loss of competent vessels, impairment of vascular regulatory mechanisms, and ultimately as the 'no-reflow' phenomenon (inability to sufficiently reperfuse previously ischaemic tissue despite the removal of the initial obstruction or occlusion). It is now appreciated that the earliest distinguishing feature of various forms of vascular injury (including atherosclerosis and infarction) is 'endothelial dysfunction', which is the marked reduction in endothelial-dependent relaxation due to reduced release or action of endothelial nitric oxide (NO). Importantly, vascular injury may worsen myocardial damage in vivo [4,5], significantly limiting tissue salvage and recovery. The pathogenesis of post-ischaemic vascular injury and endothelial dysfunction is incompletely understood, but has generally been considered to reflect a cardiovascular inflammatory response, neutrophils playing a key role. However, while blood-borne cells and inflammatory elements are undoubtedly involved in the 'progression' of vascular injury [6,7], accumulating evidence indicates that they are not the primary 'instigators' [8]. It should be noted that a wealth of controversial findings exists in the vascular injury literature and mechanisms involved remain unclear. Indeed, multiple mechanisms are likely to contribute to post-ischaemic vascular injury. Adenosine receptors are unique in playing a role in physical regulation of coronary function, and also in attenuating injury during and following ischaemia. While the adenosine receptor system has been extensively investigated in terms of effects on myocardial injury [9,10], little is known regarding potential effects of this receptor system on post-ischaemic coronary vascular injury. This thesis initially attempts to further our understanding of the role of adenosine in normal coronary vascular function, subsequent chapters assess the effect of ischaemia-reperfusion on vascular function, and adenosine receptor modification of vascular dysfunction in the isolated asanguinous mouse heart. Specifically, in Chapter 3 the receptor subtype and mechanisms involved in adenosine-receptor mediated coronary vasodilation were assessed in Langendorff perfused mouse and rat hearts. The study revealed that A2A adenosine receptors (A2AARs) mediate coronary dilation in the mouse vs. A2B adenosine receptors (A2BARs) in rat. Furthermore, responses in mouse involve a sensitive endothelial-dependent (NO-dependent) response and NO-independent (KATP-mediated) dilation. Interestingly, the ATP-sensitive potassium channel component predominates over NO-dependent dilation at moderate to high agonist levels. However, the high-sensitivity NO-dependent response may play an important role under physiological conditions when adenosine concentrations and the level of A2AAR activation are low. In Chapter 4 the mechanisms regulating coronary tone under basal conditions and during reactive hyperaemic responses were assessed in Langendorff perfused mouse hearts. The data support a primary role for KATP channels and NO in mediating sustained elevations in flow, irrespective of occlusion duration (5-40 s). However, KATP channels are of primary importance in mediating initial flow adjustments after brief (5-10 s) occlusions, while KATP (and NO) independent processes are increasingly important with longer (20-40 s) occlusion. Evidence is also presented for compensatory changes in KATP and/or NO mediated dilation when one pathway is blocked, and for a modest role for A2AARs in reactive hyperaemia. In Chapter 5 the impact of ischaemia-reperfusion on coronary function was examined, and the role of A1 adenosine receptor (A1AR) activation by endogenous adenosine in modifying post-ischaemic vascular function was assessed in isolated buffer perfused mouse hearts. The results demonstrate that ischaemia does modify vascular control and signficantly impairs coronary endothelial dilation in a model devoid of blood cells. Additionally, the data indicate that post-ischaemic reflow is significantly determined by A2AAR activation by endogenous adenosine, and that A1AR activation by endogenous adenosine protects against this model of vascular injury. Following from Chapter 5, the potential of A1, A2A and A3AR activation by exogenous and endogenous agonists to modulate post-ischaemic vascular dysfunction was examined in Chapter 6. Furthermore, potential mechanisms involved injury and protection were assessed by comparing effects of adenosine receptors to other 'vasoprotective' interventions, including anti-oxidant treatment, Na+/H+ exchange (NHE) inhibition, endothelin (ET) antagonism, and 2,3-butanedione monoxime (BDM). The data acquired confirm that post-ischaemic endothelial dysfunction is reduced by intrinsic A1AR activation, and also that exogenous A3AR activation potently reduces vascular injury. Protection appears unrelated to inhibition of ET or oxidant stress. However, preliminary data suggest A3AR vasoprotection may share signalling with NHE inhibition. Finally, the data reveal that coronary reflow in isolated buffer perfused hearts is not a critical determinant of cardiac injury, suggesting independent injury processes in post-ischaemic myocardium vs. vasculature. Collectively, these studies show that adenosine has a significant role in regulating coronary vascular tone and reactive hyperaemic responses via NO and KATP dependent mechanisms. Ischaemia-reperfusion modifies vascular control and induces significant endothelial dysfunction in the absence of blood, implicating neutrophil independent injury processes. Endogenous adenosine affords intrinsic vasoprotection via A1AR activation, while adenosinergic therapy via exogenous A3AR activation represents a new strategy for directly protecting against post-ischaemic vascular injury.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains xviii, 121 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 99-108).

Thesis (Ph.D. in Neuroscience) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 114-131).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 139-159).

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 29-32.

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