During the past 50 years, a number of nucleophosphodiesterases have been studied. Of recent interest, is a brain phosphodiesterase which converts ribonucleoside 2',3'-cyclic phosphates to the corresponding ribonucleoside 2'-phosphates. The physiological role of this enzyme is entirely unknown. The present studies were designed to learn something of its intracellular distribution, properties and mechanism of action. The assay of the enzyme was based on the hydrolysis of adenosine 2', 3'-cyclic phosphate by the diesterase followed by the hydrolysis of the reaction product with alkaline phosphatase and subsequent analysis for inorganic phosphate by a modified method of Fiske and SubbaRow. Rabbit brain homogenate was fractionated into nuclear, mitochondrial, microsomal and 100,000 x g supernatant fluid fractions by differential centrifugation. 2',3'-Cyclic phosphodiesterase activity was found in all the fractions with the greatest activity in the mitochondrial fraction. Since the mitochondrial fraction was a heterogenous mixture as revealed by electron microscopy, its components were separated by sucrose density gradient centrifugation by established methods. Five subtractions were obtained (A, B, C, D, E). The lightest subfraction (A) contained most of the diesterase activity and electron microscopy revealed that this subfraction consisted of fragments of myelin of various sizes. The microsomal and nuclear fractions were also subjected to sucrose density gradient centrifugation. Again, the least dense, myelin-containing sub-fraction, contained most of the diesterase activity. It was also found that rabbit brain white matter contained greater diesterase activity than grey matter. The data provide strong evidence that the diesterase is associated with myelin. It was found that acetone extraction of the myelin fraction from a homogenate of white matter of beef brain resulted in a doubling of diesterase activity with a five-fold purification. Efforts directed toward solubilization of the enzyme were unsuccessful. Some properties of the enzyme were also determined. The diesterase opened the cyclic diester bond of cyclic-ended oligonucleotides, at least as large as (A₈) and did so, without cleavage of internucleotide bonds. It was inactivated by the sulfhydryl reagent, para-hydroxymercuribenzoate, and was unaffected by the presence of a wide variety of purine and pyrimidine compounds or derivatives and various nucleoside mono-, di- and triphosphates. The Km of the diesterase was determined to be 1.9 x 10⁻³ M. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

No description available.

No description available.

Damages to the genetic materials arise throughout the lifespan of a cell, and elicit upregulation of DNA repair factors. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is part of a DNA repair protein complex that specialises in the repair of DNA base modifications and single-strand breaks (SSBs). TDP1 removes a broad spectrum of chemical adducts from the 3' end of a DNA strand break, including topoisomerase 1 (TOP1) peptide, during DNA transcription and replication. Inactivation or deletion of TDP1 is associated with cerebellar dysfunction and degeneration, with remarkably little extraneurological manifestation. The reason for the selective dependence of the cerebellar neurons on TDP1 activity is not clear. It was hypothesised that the TDP1 activity is upregulated in tissues with high levels of SSBs, either from DNA transcriptional activity, or reactive oxygen species (ROS)-induced damage. The aim of this doctoral project was therefore to identify and characterise the cellular mechanisms that regulate TDP1 activity. Our lab has previously shown that the Nterminus domain (NTD) of TDP1 covalently interacts with DNA ligase 3α. In this thesis, evidence has been presented to show that this interaction is regulated by the putative ATM/ATR/DNA-PK phosphorylation site, serine 81, to prolong TDP1 half-life, and enhance cellular survival after genotoxic stress. A second post-translational modification in the NTD by SUMOylation of the K111 residue was identified, enlightening a mechanism by which TDP1 is recruited to sites of transcription-mediated SSBs. To investigate the requirement for TDP1 in cells under high levels of oxidative stress, I have developed a mouse cellular model whereby the levels of endogenous ROS can be modulated by overexpression of the human anti-oxidant enzyme superoxide dismutase 1 (SOD1) or its toxic mutant SOD1G93A. Overexpression of SOD1G93A in Tdp1-/- MEFs induces accumulation of chromosomal SSBs and decreases survival after H2O2 challenge, while overexpression of SOD1 has a protective effect. Besides repair of ROS-induced TOP1-cc in the nucleus, TDP1 also repairs mitochondrial topoisomerase 1-mediated DNA breaks. This role is required during transcription and assembly of mitochondrial subunits of the electron transfer chain complexes, and has direct impact on mitochondrial respiration and ROS production. Collectively, these data provide mechanistic insights into regulation of TDP1-mediated chromosomal and mitochondrial DNA repair.

PART I STUDIES ON HEART MUSCLE LIPASES The study of the role of lipids in supplying the energy requirements of the heart has attracted widespread attention, particularly within the past decade. It is now known that the heart, under normal conditions, oxidizes lipids as its main source of energy. Numerous investigators have studied the in vivo and in vitro uptake and utilization of exogenously supplied lipids in the form of triglycerides, free fatty acids and ketone bodies. However, very few have studied the utilization of endogenous lipids by the working heart. We have examined the relative importance of both endogenous glycogen and triglycerides for supplying the caloric needs of the isolated beating rat heart, and found that under the perfusion conditions used, endogenous glycogen appears to supply the initial source of energy. A lipase in rat cardiac tissue was also examined. The enzyme had a pH optimum near 6.8, and was strongly inhibited by 0.2 M NaF and by 2 x 10⁻⁴M diisopropylfluorophosphate. Most of the activity was found in the nuclear fraction of tissue homogenates. The enzyme hydrolyzed both monoolein and mono-stearin, and possessed much less activity against tripalmitin. The enzyme also rapidly hydrolyzed the monostearin component of Ediolʀ (a commercial coconut oil emulsion widely used in lipase studies), and the implications of these findings are discussed. It was concluded from these studies that a lipase other than lipoprotein lipase exists in rat myocardium. PART II STUDIES ON CYCLIC 3', 5'-NUCLEOTIDE PHOSPHODIESTERASE In recent years, the study of the role of cyclic 3', 5'-adenosine monophosphate (cyclic 3', 5'-AMP) in the regulation of several biological reactions and processes has received widespread attention. The presence of a physiological mechanism for terminating the action of cyclic 3', 5'-AMP in biological systems would therefore be expected. Indeed, an enzyme, cyclic 3', 5'-nucleotide phosphodiesterase has been shown to exist in most mammalian tissues which have been studied for its activity. The central nervous system, particularly the cerebral cortex, possesses a very high activity of this enzyme. In this study, cyclic 3', 5'-nucleotide phosphodiesterase was partially purified from rabbit brain and its properties were studied. The enzyme required Mg⁺⁺ions for activity and was inhibited by 2 x l0⁻⁴M theophylline. Cyclic 3', 5'-dAMP, cyclic 3', 5'-GMP and cyclic 3', 5'-dGMP were hydrolyzed by the brain diesterase at approximately one-half the rate at which cyclic 3', 5'-AMP was hydrolyzed. Little activity against cyclic 3', 5'-CMP, cyclic 3', 5'-dCMP and cyclic 3', 5'-TMP was detected, although cyclic 3', 5'-UMP was hydrolyzed at approximately 13% of the rate at which cyclic 3', 5'-AMP was hydrolyzed. The brain diesterase therefore possessed a high specificity for cyclic 3', 5'-nucleotides with purine bases. Optimum enzyme activity was observed near pH 7.0, and the activity was stimulated about 1.5-fold by 0.06 M imidazole. The Km value of the enzyme with cyclic 3', 5'-AMP as substrate was approximately 0.8 x 10⁻⁴M. The properties of the partially purified phosphodiesterase from brain were thus very similar to the diesterases which have been purified from beef and dog hearts. A study of the intracellular localization of the brain diesterase indicated that about 50% of the activity was located in the 105,000 x g supernate. The microsomal and mitochondrial fractions also contained considerable amounts of diesterase activity, but little activity was located in the nuclear fraction. A survey of cyclic 3', 5'-nucleotide phosphodiesterase activity in several available specimens of the plant kingdom indicated the absence of this enzyme activity in these organisms. However, appreciable levels of diesterase activity were detected in E. coli. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

The distribution and some of the properties of phosphodiesterase II were studied in homogenates of rat intestinal mucosa in an attempt to elucidate its role in the nucleic acid metabolism of this tissue. In most of the experiments the p-nitrophenyl ester of thymidine 3'-phosphate was used as a substrate for phosphodiesterase. During the work, evidence was accumulated which indicated that phosphodiesterase II of intestine was lysosomal in origin. For instance, when the tissue was suspended (or homogenized) in media of differing tonicity, the phosphodiesterase II activity in the hypotonic preparations increased markedly over a period of 96 hours. Other investigators have shown that this "osmotic activation" is a characteristic of lysosomal enzymes. Subsequently, homogenates of mucosal tissue were fractionated by differential centrifugation and the subcellular fractions obtained were identified by known enzyme markers. The distribution of phosphodiesterase II in the fractions was most similar to that of the marker for lysosomes - acid phosphatase. However a large proportion of the phosphodiesterase II activity, greater than that of acid phosphatase, was found in the supernatant solution remaining after the final high-speed centrifugation step. The highest specific activity for phosphodiesterase II was found in the "light mitochondrial" and "final supernatant" fractions. Similar results were obtained when homogenates or nuclei-free homogenates were fractionated by sucrose density-gradient centrifugation. The distribution patterns of phosphodiesterase II and acid phosphatase were again similar and the particles to which phosphodiesterase II were bound exhibited the highest acid phosphatase activity. An attempt was made to confirm these results by "purifying" lysosomes from intestinal mucosa using a combined differential centrifugation and density-gradient centrifugation technique. During the purification, the specific activities of phosphodiesterase II and acid phosphatase increased parallel with each other and the "purified lysosomal" fractions exhibited the highest specific activities for these enzymes. However the total activities of the two enzymes recovered in the purified fractions were quite small, indicating considerable loss in the discarded soluble fractions. Other workers have shown that homogenization ruptures lysosomes in certain fragile tissues, resulting in high soluble activities of the enzymes contained in these particles. It would seem possible therefore that in intestinal mucosa phosphodiesterase II is located in lysosomes in vivo, since most of its activity was found to be distributed between the lysosomal and soluble fractions of homogenates of this tissue. The phosphodiesterase II of intestine was most active at pH values around neutrality. A second substrate, 2,4-dinitro-phenyl thymidine 3'-phosphate, which was used in only"a few experiments because of its limited availability, was hydrolyzed at a rate faster than that of p-nitrophenyl thymidine 3'-phosphate. Little or no change in the activity of the enzyme was observed in the presence of Mg++, Ca++ or EDTA, but Zn++, Cu++ and Hg++ inhibited markedly. The enzyme was most active at a temperature of 58° and only 27% of the activity was lost on heating the preparation for 1 hour at 55°. The Michaelis constant for the enzyme with p-nitrophenyl thymidine 3'-phosphate was 4.5 x 10(-4) M at 37°, and the activation energy for the phosphodiesterase II catalyzed hydrolysis of the same compound was 14.63 kilocalories/ mole. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Cyclic nucleotides are important inhibitory regulators of platelet function. These second messengers are hydrolysed by cyclic 3' ,5'-nucleotide phosphodiesterases (PDEs). Three PDEs have been detected in human platelets: cGMP-stimulated phosphodiesterase (PDE2), cGMP-inhibited phosphodiesterase (PDE3), and cGMP-binding, cGMP-selective phosphodiesterase (PDE5). This research investigates the contribution of PDE2 to the regulation of platelet cyclic nucleotide concentrations, and the effects that PDE2 activity has on the inhibition by cAMP and cGMP of platelet aggregation in response to thrombin or collagen. Increases in platelet cAMP were initiated by stimulation of adenylyl cyclase with prostacyclin (PGI₂), whereas the accumulation of cGMP was induced by nitroprusside (NP). The contributions of PDE2 to the hydrolysis of these cyclic nucleotides were evaluated using a novel inhibitor of the enzyme, 𝘦𝘳𝘺𝘵𝘩𝘳𝘰-9-(2-hydroxy-3-nonyl)adenine (EHNA). Before EHNA was used in experiments on platelet function, its effects on partially purified preparations of the three platelet PDEs were studied. These investigations demonstrated that EHNA is a selective and potent inhibitor of platelet PDE2, and indicated that this compound is a more effective inhibitor of cAMP hydrolysis in the presence than in the absence of cGMP. To measure changes in cyclic nucleotide concentrations, platelets were preincubated with [³H]adenine and ³H]guanine to label the metabolic nucleotide pools. NP caused large concentration-dependent increases in platelet [³H] cGMP levels, and this was associated with highly significant but much smaller increases in [³H] cAMP accumulation, which were optimal with 10 μM NP. Higher concentrations of NP had much less effect on platelet [³H] cAMP. A previous study had shown that the increases in platelet cAMP caused by NP were attributable to the inhibition of PDE3 by cGMP (Maurice and Haslam, 1990a), but the inhibitory component observed with high concentrations of NP had not been explained. The present research showed that the accumulation of cAMP and cGMP induced by high NP concentrations is enhanced by EHNA, and so provides the first demonstration that PDE2 activity restricts NP-induced cyclic nucleotide accumulation. To assess whether these changes in platelet cyclic nucleotide levels were important, platelet aggregation in response to thrombin and collagen was monitored. In these studies, EHNA markedly increased the inhibitory action of NP on platelet aggregation. All the effects of NP on cyclic nucleotide accumulation and on platelet aggregation were blocked by a guanylyl cyclase inhibitor, 1𝘏-[1,2,4] oxadiazolo [4,3-α] quinoxalin-1-one, confirming that NP acts solely through activation of this enzyme and that the increases in cAMP are secondary to cGMP formation. However, experiments with the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine, which diminished the accumulation of cAMP but not that of cGMP, indicated that the inhibition of platelet aggregation is more closely correlated with the increases in cAMP than with those in cGMP. In experiments in which platelet PDE3 was selectively blocked by lixazinone, the accumulation of [³H]cAMP was greatly increased and a corresponding inhibition of thrombin-induced platelet aggregation was observed. Both of these effects were greatly diminished when PDE2 was stimulated by NP (or cGMP). This research demonstrates for the first time that activation of PDE2 by cGMP has marked effects on platelet function, restricting the inhibition of platelet aggregation by agents that increase platelet cAMP. To investigate the importance of PDE2 in regulating different platelet cAMP levels, the effects of EHNA were studied in the presence of 1 or 20 nM PGI₂. Whereas no significant increase in cAMP accumulation was caused by EHNA in the presence of 1 nM PGI₂, at the higher PGI₂ concentration a marked increase was detected when PDE2 was inhibited. NP potentiated the increase in cAMP seen with low PGI₂ but inhibited that seen with a high PGI₂ concentration, indicating a shift in the relative importance of PDE3 and PDE2 as platelet cAMP was increased. These studies show that in the presence of a high concentration of cAMP alone, or of regulatory cGMP, PDE2 makes a major contribution to the hydrolysis of platelet cAMP. Moreover, the results suggest that PDE2 inhibitors could be of value in the therapeutic modification of platelet responses. / Thesis / Master of Science (MSc)

No description available.

Heart failure is the leading cause of morbidity and mortality in the world. It is an incurable disease and most treatment strategies aim to treat the symptoms or slow the progression of the condition. Cardiac contractility is governed by calcium homeostasis within cardiac myocytes and is modulated by the sympathetic nervous system. Both mechanisms are detrimentally altered in heart failure. An important group of enzymes, phosphodiesterases, are fundamental to the sympathetic (beta-adrenergic) modulation of calcium cycling in cardiac myocytes. The selective inhibition of phosphodiesterase 5 (PDE5) has recently been considered as a potential therapy for heart failure; having beneficial effects in human and animal models of the disease. The present study employs a large animal model of tachypacing induced heart failure to test the effect of PDE5 inhibition on myocyte and whole heart contractility and beta-adrenergic function, to assess the molecular mechanisms by which PDE5 inhibition is beneficial to the failing myocardium. In initial experiments the PDE5 inhibitor sildenafil was applied acutely to voltage clamped ventricular myocytes from uninstrumented sheep. PDE5 inhibition reduced baseline L-type calcium current and systolic calcium transient amplitude, suggesting it is negatively inotropic. Furthermore, the positive inotropic effects of beta-adrenergic stimulation were somewhat reversed by acute PDE5 inhibition. Interestingly, such negative inotropic effects of acute PDE5 inhibition were not observed in failing ventricular myocytes, which have dysfunctional calcium homeostasis and beta-adrenergic reserve. When delivered chronically over 3 weeks to tachypaced animals, PDE5 inhibition restored and augmented the systolic calcium transient and beta-adrenergic responsiveness at both the whole heart and myocyte level. These effects were associated with changes to the expression and phosphorylation status of the proteins that control calcium homeostasis in left ventricular tissue. In vivo, PDE5 inhibition prolonged longevity and reduced the onset of clinical signs of heart failure in sheep, as well as arresting cardiac dilatation and wall thinning. Chronic PDE5 inhibition however had no effect on cardiac contractility or heart failure induced changes in cardiac electrophysiology. This study presents a novel mechanism by which PDE5 inhibition may be beneficial in a large animal model of heart failure by restoring calcium homeostasis and beta-adrenergic responsiveness. This study may have important implications for the management of heart failure in clinical practice.

Thesis (Ph.D)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Physiology. Title from title-page of electronic thesis. Includes bibliographical references.