No description available.

The cytoplasmic enzyme, NAD⁺ kinase (ATP: NAD⁺ 2-phosphotransferase, [E.C. 2.7.1.23}) has been characterized and purified from yeast. A continuous fluorescence assay was developed. A purification procedure was developed utilizing NAD⁺-Agarose affinity column chromatography.

We conducted 16 parallel in vitro selection experiments to isolate catalytic DNAs from a common DNA library for the cleavage of all 16 possible dinucleotide junctions of RNA incorporated into a common DNA/RNA chimeric substrate sequence. We discovered hundreds of sequence variations of the 8-17 deoxyribozyme - an RNA-cleaving catalytic DNA motif previously reported - from nearly all 16 final pools. Sequence analyses identified four absolutely conserved nucleotides in 8-17. Five representative 8-17 variants were tested for substrate cleavage in trans and together they were able to cleave 14 dinucleotide junctions. New 8-17 variants required Mn2+ to support their broad dinucleotide cleavage capabilities. We hypothesize that 8-17 has a tertiary structure composed of an enzymatic core executing catalysis and a structural facilitator providing structural fine-tuning when different dinucleotide junctions are given as cleavage sites. / Thesis / Master of Science (MSc)

The levels of total nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), and their redox states were determined as the function of growth in S. cerevisiae. Cells growing in a medium containing 0.8% glucose exhibit two phases of exponential growth, utilizing glucose and ethanol, respectively. The NAD pool is 50% reduced during both stages of growth while the NADP pool is 67% reduced in glucose growth and 48% reduced in ethanol growth. The NAD/NADP ratio is constant during growth on glucose and a two-fold increase in the NAD/NADP ratio occurs upon exhaustion of glucose. The increased ratio is maintained during growth on ethanol. This alteration in the regulation of the relative levels of NAD and NADP may be due to a change in the regulation of NAD kinase and/or NADP phosphatase activities. These changes may be related to the redox state of the NADP pool.

A protein exhibiting NADH oxidase and NADH peroxidase activity has been isolated from Lactobacillus casei in high purity. Evidence obtained through gel filtration, electrophoresis, and electrofocusing indicates that the two activities are characteristic of a single tetrameric protein with an approximate molecular weight of 240,000.

Nicotinamide adenine dinucleotide (NAD+) is a cofactor in hydride transfer reactions and consumed substrate of several classes of glycohydrolyitc enzymes, including sirtuins. NAD+, its biosynthetic intermediates, breakdown products, and related nucleotides (the NAD metabolome) is altered in many metabolic disorders, such as aging and obesity. Supplementation with the novel NAD+ precursor, nicotinamide riboside (NR), ameliorates these alterations and opposes systemic metabolic dysfunctions in rodent models. Based on the hypothesis that perturbations of the NAD metabolome are both a symptom and cause of metabolic disease, accurate assessment of the abundance of these metabolites is expected to provide insight into the biology of diseases and the mechanism of action of NR in promoting metabolic health. Current quantitative methods, such as HPLC, lack specificity and sensitivity to detect distinct alterations to the NAD metabolome. In this thesis, I developed novel sensitive, accurate, robust liquid chromatography mass spectrometry methodologies to quantify the NAD metabolome and applied these methods to determine the effects of disease states and NR supplementation on NAD+ metabolism. My investigations indicate that NR robustly increases the NAD metabolome, especially NAD+ in a manner kinetically different than any other NAD+ precursor. I provide the first evidence of effective NAD+ supplementation from NR in a healthy, 52 year old human male, suggesting the metabolic promoting qualities of NR uncovered in rodent studies are translatable to humans. During my investigation of NR supplementation, my work establishes an unexpected robust, dramatic increase in deamino–NAD+, NAAD, directly from NR, which I argue could serve as an accessible biomarker for efficacious NAD+ supplementation and the effect of disease upon the NAD metabolome. Lastly, I further establish NR as a general therapeutic against metabolic disorder by detailing its ability to oppose aspects of chronic alcoholism and diabetes mellitus.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 105-118).

Thesis (doctoral)--Rijksuniversiteit te Groningen. / Description based on print version record.

Poly(ADP-ribosyl)ation is a reversible post-translational modification of histones and nuclear proteins rapidly stimulated by DNA damage. Its homeostasis is a dynamic process regulated by the synthesizing enzymes poly(ADP-ribose) polymerases (PARPs) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). PARP-1, the first-discovered and major PARP, has been the focus of many studies aimed at clarifying the biological function of poly(ADP-ribose) (PAR). This abundant nuclear enzyme plays key roles in a variety of cellular processes, including the regulation of chromatin structure, transcription and genomic integrity. Its multifunctionality has made it an attractive and potential target for therapy, as evidenced by the numerous PARP-1 inhibitors currently undergoing clinical trials. The transient nature of PAR, explained by the close coordination between PARP-1 and PARG, has also highlighted the potential of targeting PARG for diseases of inappropriate cell death. A number of obstacles, however, have prevented PARG from being studied as extensively as PARP-1. The extreme sensitivity of PARG to proteases and its insolubility at high concentrations have limited structure-activity relationship analyses and structural studies of PARG, and the unavailability of high-throughput activity assays has stalled the discovery and development of specific and cell permeable PARG inhibitors, subsequently slowing down the validation of PARG as a therapeutic target. The work presented in this dissertation describes in detail strategies devised to overcome these difficulties. First, a novel colorimetric high-throughput assay for PARG was evaluated and its sensitivity and precision were compared to a widely-used radiolabelling assay. Second, several expression and purification systems were constructed in order to obtain high quantities of soluble human PARG protein adequate for in vitrostructural studies. The efficacy of these strategies was demonstrated in structure-activity analyses of PARG which led to the identification of a regulatory segment far removed linearly from the catalytic site of PARG. This region, necessary for catalytic activity, corresponds with a recently identified mitochondrial targeting sequence (MTS) and was thus named the ‘regulatory segment/MTS’ (REG/MTS). Finally, based on structural data obtained, secondary structure predictions were made to provide insight into the molecular composition of the different domains of PARG, whose structures still remain to be determined.

Novel Ca²⁺ signalling pathways in both endothelial cells and smooth muscle cells of rat small resistance arteries were investigated using a combination of confocal imaging, isometric tension recordings, and electrophysiology to study freshly isolated arteries and cells. We first examined the hypothesis that hyperpolarization could alter endothelial cell Ca²⁺ events. Hyperpolarization evoked by direct opening of K_ATP channels in the smooth muscle with levcromakalim triggered an increase in the frequency of Ca²⁺ events in the endothelium of rat cremaster arterioles. These Ca²⁺ events were discrete in nature, requiring subcellular regions of interest to reliably identify them. Opening of K_ATP channels indirectly through β-adrenoceptor stimulation with isoprenaline, caused a similar increase in the frequency of endothelial cell Ca²⁺ events in rat mesenteric third order arteries. These events also had a similar, focal profile. Pharmacological investigation suggested that the response to isoprenaline was receptor-mediated, and dependent on Ca²⁺ influx and opening of K_ATP channels. The presence of β-adrenoceptors on endothelial cells was confirmed using fluorescently-tagged β-adrenoceptor ligands, which showed punctate labelling in smooth muscle and endothelial cells of rat mesenteric arteries. Freshly isolated endothelial cells also showed Ca²⁺ increases to isoprenaline, although this was not consistently observed. Following on from the observed endothelial cell Ca²⁺ response to hyperpolarization, we tested the hypothesized involvement of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels using the channel inhibitor, ZD7288. Pre-treatment with ZD7288 (1 μM) reduced both the endothelial cell Ca²⁺ response to isoprenaline (in mesenteric arteries) and levcromakalim (in cremaster arterioles). HCN channel subtypes were identified in cremaster arterioles through immunolabelling. We also observed an interesting effect of higher concentrations of ZD7288 to potentially inhibit K⁺ channels, including endothelial cell KCa channels, since hyperpolarization to isoprenaline, levcromakalim or acetylcholine (ACh) was reduced by 10 μM ZD7288, and relaxation to ACh was partially inhibited. ACh-mediated relaxation was also partially inhibited by the clinically used HCN channel blocker, ivabradine (0.3-30 μM). Finally, we identified an interaction of the Ca²⁺-releasing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) with BKCa channels in the smooth muscle. NAADP-mobilised Ca²⁺ has been reported to interact with ryanodine receptors hence we hypothesized an interaction with BK_Ca channels via Ca²⁺ sparks. We found that NAADP-AM relaxed and hyperpolarized rat mesenteric arteries, which was blocked by iberiotoxin (BK_Ca channel inhibitor) and high extracellular [K⁺] (45 mM). Furthermore, NAADP increased paxilline-sensitive K⁺ currents and the frequency and amplitude of spontaneous transient outward currents (STOCs) in freshly isolated vascular smooth muscle cells patched in the whole-cell configuration, further supporting an action at BK_Ca channels. All together these data identify novel Ca²⁺ signalling pathways in resistance arteries that are both activated by and promote hyperpolarization, which is a key determinant of vascular tone.