No description available.

No description available.

Atherosclerosis is a chronic inflammatory disease leading to plaque buildup in the major arteries. The plaques consist of cholesterol, calcium, inflammatory cells, extracellular matrix and fibrous material. Under inflammatory conditions IFN-• stimulation of human monocytes and macrophages generates reduced pteridine, 7,8-dihydroneopterin (78NP) which has been shown to be an effective cytoprotective agent to some cell types against oxidative damage by reactive oxygen species (ROS). 7,8-dihydroneopterin is oxidized to fluorescent neopterin in the presence of hypochlorite (HOCl). Although a considerable amount of work has been published on the composition of neopterin in atherosclerotic plaques, very little is known about the variation of 78NP and other oxidative biomarkers across the length of the carotid and femoral and their contribution to plaque progression, which was researched in this work. Atherosclerotic plaques excised from patients with carotid and femoral plaques were sliced into 3-5 mm sections, and each section was analyzed for concentrations of neopterin, 7,8- dihydroneopterin, •-tocopherol, TBARS, DOPA, cholesterol, dityrosine, protein carbonyls •- aminoadipic semialdehyde (AAS) and •-glutamic semialdehyde (GGS), free and esterified 7- ketocholesterol (7-KC). Cultured live plaque as a source of 7,8-dihydroneopterin and neopterin was also investigated in this study. It was shown that carotid plaques significantly vary from femoral plaques, in the levels and range of most oxidative biomarkers. Carotid plaques showed a high variation in the biomarker concentrations between plaques but also between sections of an individual plaque. Femoral plaques on the other hand showed lower amounts of biomarkers with very little variation in biomarker concentrations. High variation with pterin concentrations and other biomarkers suggests dynamic and active changes in inflammation within the plaque. Collectively, it was observed that every plaque was unique with respect to its composition and correlations between the biomarkers. Though shown to be a well-known antioxidant and a radical scavenger, there is no published literature on 7,8-dihydroneopterin’s mode of entry into and out of the cell. To understand how it enters the cells could explain the difference in its protective ability of different cell types Abstract xxviii against oxidative stress-mediated cell death. Knowledge of transport of 7,8-dihydroneopterin will provide insights about its protection of monocyte/macrophage cell death which could potentially reduce atherosclerotic plaque growth and progression. As 7,8-dihydroneopterin is produced from guanosine, a nucleoside that is transported using specialized nucleoside transporters (equilibrative nucleoside transporters (ENT's) and concentrative nucleoside transporters (CNT's), their role was examined and characterized for 7,8-dihydroneopterin transport. It was found that 7,8-dihydroneopterin and neopterin are transported via nucleoside transporters in U937 cells, THP-1 cells and human monocytes. ENT 2 was the major transporter in U937 cells while ENT 1 transported bulk of 7,8-dihydroneopterin in THP-1 cells. Both ENT's and CNT's are involved in 7,8-dihydroneopterin uptake in human monocytes. In all the cell lines tested, 7,8-dihydroneopterin protection against AAPH mediated oxidative cell death was inhibited by nucleoside transport inhibitors, suggesting that nucleoside transporters are indispensible for 7,8-dihydroneopterin mediated intracellular protection against oxidative stress. Accurate measurement of neopterin, as a biomarker of inflammation in plaques and cells is critical aspect to assess disease progression. The current C18 HPLC method used in our laboratory for neopterin measurement lacks sensitivity due to interference of acetonitrile (ACN) over time. Acidic tri-iodide conversion of 7,8-dihydroneopterin to neopterin was also variable at times giving inconsistent measurement of neopterin so the manganese oxide (MnO2) method was looked at as an alternative. Electrochemical detector (ECD) was another option studied as it did not require any precolumn oxidation of 7,8-dihydroneopterin to neopterin. A new method using strong cation exchange (SCX) column was developed for a precise, sensitive neopterin assay which got rid of the ACN interference completely. The MnO2 method of 7,8-dihydroneopterin oxidation did not work with biological samples such as serum or plaque homogenates. Electrochemical detection was also found to be very unreliable and inconsistent.

Age-related macular degeneration (AMD) is a principal cause of blindness in the United States and other industrialized nations. An estimated 10 million Americans are afflicted with AMD, which is comparable in scope to the 12 million living with cancer, or the 5 million with Alzheimer’s disease. The prevalence of AMD steadily increases with age, affecting 2% of the population at age 40, and one in four people by age 80. For reasons that are not fully understood, AMD is more common in lightly-pigmented and female populations. Treatment of AMD is largely an unmet need: There are no FDA approved therapies except for a small percentage of individuals with end-stage disease. This dissertation investigates the mechanisms of AMD pathogenesis and offers insight into novel therapeutic strategies for this disease.

Several nucleoside natural product antibiotics from Streptomyces sp. and actinomycetes have recently been shown to target bacterial peptidoglycan cell wall biosynthesis by inhibiting the bacterial translocase I (MraY). The biosynthetic gene clusters for A-90289, liposidomycins and caprazamycins revealed a protein with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD). This enzyme (LipL) is a mononuclear, non-heme, Fe(II) dependent α-keto glutarate (α-KG) :uridine monophosphate (UMP) dioxygenase responsible for the net dephosphorylation and two electron oxidation of UMP to uridine-5’-aldehyde. The postulated reaction coordinates involving the activation of the C-5’ center in UMP and the corresponding formation of uridine-5’-aldehyde are modeled on extensive spectroscopic and structural characterizations of TauD. In this dissertation, the postulated radical mechanism for LipL involving the formation of an unstable hydroxylated intermediate is investigated via the characterization of a key product obtained from the reaction of LipL (and its homolog Cpr19) with a synthetically modified surrogate substrate where the bridging phosphoester oxygen in UMP is replaced with a 5’ C-P bond. We further validate our hypothesis by analyzing the reactions of both LipL and Cpr19 with specifically 2H1 – labeled UMP substrate and confirming the expected products via mass spectrometry. In addition, we explore substrate promiscuity of the enzymes and utilize a set of site specific mutants of Cpr19 as means of gaining better insight into the active site residues. Predictive models for Cpr19 and LipL structures are developed by the combination of experimental results and chemical logic.

No description available.

Human cells possess multiple nucleoside transporters (NTs) that belong to either the human equilibrative or concentrative NT (hENT: hENT1/2/3/4; hCNT: CNT1/2/3) families. In the kidney, coupling of apical hCNT3 activities to basolateral hENT1/2 activities is hypothesized to mediate renal nucleoside proximal tubular absorption while apical ENT1 may have a role in secretion. The overall aim of this research was to increase understanding of the roles of hENTs and hCNTs in renal handling of physiological nucleosides and anti-cancer nucleoside analog drugs. This was achieved by investigating the distribution of hENTs and hCNTs in human kidney tissue and the function of hENTs and hCNTs in cellular uptake and transepithelial fluxes of nucleosides in cultured human renal proximal tubule cells (hRPTCs). Immunolocalization of hCNT3 and hENT1 in human kidney tissue revealed that hENT and hCNT3 were present in apical membranes of proximal tubules. Production and characterization of adherent hRPTC cultures demonstrated endogenous hCNT3, hENT1, and hENT2 activities. These results provided evidence for the involvement of hCNT3, hENT1, and hENT2 in renal handling of nucleosides. Comparison of adherent hRPTC cultures derived from kidneys from different individuals demonstrated that hCNT3 activities varied between cultures. Also, the extent of cellular uptake of fludarabine, an anti-cancer nucleoside drug, and degree of cytotoxicity was reflected in the different hCNT3 activities observed between cultures. These results suggested that hCNT3 plays an important role in fludarabine renal handling and is a determinant of potential renal toxicities. Production of polarized monolayer cultures of hRPTCs on transwell permeable inserts enabled the functional localization of hCNT3 and hENT1 to apical membranes and hENT2 to basolateral membranes. Transepithelial flux studies demonstrated that (i) apical-to-basolateral fluxes of adenosine were mediated by apical hCNT3 and basolateral hENT2, (ii) basolateral-to-apical fluxes of 2′-deoxyadenosine were mediated, in part, by apical hENT1 and basolateral hOATs, and (iii) apical-to-basolateral fluxes of fludarabine, cladribine, and clofarabine were mediated by apical hCNT3. These studies showed that coupling of apical hCNT3 to basolateral hENT2 mediates proximal tubular nucleoside reabsorption, that coupling of basolateral human organic anion transporters (hOATs) to apical hENT1 mediates proximal tubular nucleoside secretion, and that hCNT3 is a key determinant of fludarabine proximal tubular reabsorption and cytoxicity.

A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems. In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application. AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate. Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

München, Techn. Univ., Diss., 2004.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 115-127).