Nitric oxide synthase (NOS) catalyses the production of nitric oxide (NO). A cytochrome P450-like oxygenase, it uses two monooxygenation steps to convert L-arginine (L-arg) first to N -hydroxy-L-arginine (NOHA), a stable intermediate, and then to L-citrulline and NO. Mammalian NOSs are homodimeric enzymes. Each monomer is composed of an oxygenase domain (containing the L-arg binding site, a heme ligated by a cysteine thiolate, and a tetrahydrobiopterin (H4B)) and a reductase domain (binding NADPH, FAD, and FMN). NOS substrates are O2, L-arg, and NADPH. NADPH is the source of electrons required for oxygen activation. H4B is a vital cofactor that aids dimerisation and acts as a reducing/oxidising agent. Controversy still exists as to the final oxygenating species in the NOS mechanism, but the general reaction scheme is known. The ferric heme is reduced to the ferrous state by an electron from the reductase domain. Then oxygen binds to form the oxy-ferrous species. Then H4B donates an electron to form a peroxy-ferric species. It is likely this then forms a compound 1 (Fe(IV)+.=O) species that is the final oxygenating species. This thesis probes the mechanism of NOS to further define the mechanistic intermediates involved. The role of H4B in NO synthesis has been probed in both normal turnover conditions and special case reactions. To elucidate this mechanism further a mutant with a residue capable of stabilising the activated oxygen species was created, G586S, where glycine 586 of nNOS was replaced with a serine. This serine was within hydrogen bonding distance of the oxy-heme. A stabilised intermediate was observed by stopped flow reaction in the presence of H4B, but not aH4B (an inactive pterin analogue). Here single turnover reactions, each following either the reaction of L-arg to NOHA or NOHA to citrulline, were performed on the mutant using an external source of electrons. The reaction products were observed by HPLC. The mutant appears capable of the conversion of NOHA to citrulline, but not L-arg to NOHA. The WT enzyme appears capable of both. The intermediate is observed with either L-arg or NOHA bound, suggesting both reactions proceed via the same active oxygenating species. The inability of the mutant to catalyse the conversion of L-arg to NOHA may be due to protonation of the substrate hindering reaction such that the active oxygenating species decays before reaction can occur. This mutation, in allowing separation of the two monooxygenation steps, deserves further study. H4B binds at the dimer interface of NOS. Here the -systems of the pterins are only 13Å apart. This is within allowed distances for efficient electron transfer. Electron transfer between hemes, via the pterins, would allow a route for the breakdown of a dead end, ferrous-NO, species. Stopped flow monitoring of the decay of the ferrous-heme NO complex with nNOSoxy dimers with varying proportions of the hemes in the ferrous heme-NO complex showed no electron transfer between hemes of the dimer. The rate of decay of the ferrous heme-NO complex in oxygenated buffer is 0.12 s-1 for all conditions tested here. H4B-deficiency leads to several diseases. H4B makes a poor drug due to instability and cost, the search for druggable analogues of it is ongoing. H4B analogues blocked at the 6,7-positions in the dihydropterine-form have been screened here for catalytic activity. Several have shown comparable ability to catalyse NO production in vitro. Structure function analysis of these analogues has revealed the extent extension is tolerated at the C6 and C7 positions of the pterin.

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La découverte récente de nouveaux gènes, de mécanismes d’épissage alternatif et d’interactions entre les protéines du métabolisme intracellulaire de la Cbl suggère que de nouvelles interactions protéiques peuvent prendre part aux mécanismes de régulation de ce métabolisme. Nos données confirment, dans les cellules humaines des HepG2 et des fibroblastes de patients CblC et cblG, diverses interactions qui ont été jusqu'à présent que décrites in vitro ou chez les bactéries : MS avec méthionine synthase réductase (MSR), MS avec MMACHC et MMACHC avec MMADHC. Nos données révèlent également de nouvelles interactions : MMADHC avec MS, MMADHC avec MSR, MSR avec MMACHC et MS avec les isoformes de MAT. De plus, l'absence de MS ou MMACHC perturbe les interactions impliquant les autres partenaires protéiques de l'interactome MS. En conclusion, cette étude suggère que les différentes étapes du métabolisme intracellulaire de Cbl pourraient se produire dans un grand complexe multiprotéique composé d'au moins de MS, MSR, MMACHC, MMADHC et les isoformes de MAT, et qui contribuerait à protéger la Cbl du milieu cytoplasmique / The recent discovery of new genes, alternative splicing and protein-protein interactions between intracellular processing of vitamin B12 or cobalamin (Cbl) highlights the importance of an MS interactome. The goal of this PhD project is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. Our data confirm for the first time in human cells (HepG2 cells and fibroblasts from cblC and cblG patients) various interactions that were so far only described in vitro or in bacteria: MS with methionine synthase reductase (MSR), MS with MMACHC, and MMACHC with MMADHC. Our data also reveal novel interactions: MMADHC with MTR, MMADHC with MSR, MSR with MMACHC and MS with MAT isoforms. Moreover, our data show that the absence of MS or MMACHC disturbs the interactions involving the other members of the MS interactome. In summary, this study suggests that different steps of the intracellular processing of Cbl could occur in a large multiprotein complex composed of at least MS, MSR, MMACHC, MMADHC and MAT isoforms that would contribute to protect the rare and highly reactive Cbl from the cytoplasmic milieu

Diese Arbeit zeigt, dass die spezifische Inhibition der neuronalen NO-Synthase zu einer Reduktion der myokardialen Ökonomie führt. Umgekehrt lässt sich postulieren, dass die Produktion von Stickstoffmonoxid durch die nNOS eine gesteigerte Effizienz des Herzmuskelgewebes bewirkt. / This study shows that specific inhibition of neuronal NO synthase leads to a reduction of myokardial efficiency. Vice versa one can postulate that the production of nitric oxide by nNOS improves the efficiency of the myokardium.

Lichens produce a diverse array of bioactive secondary metabolites, many of which are unique to the organisms. Their potential applications, however, are limited by their finite sources and the slow-growing nature of the organisms in both laboratory and environmental conditions. This thesis set out to investigate polyketide synthase genes in lichens, with the ultimate goal of providing a sustainable source of lichen natural products to support these applications. To expand the diversity of PKS genes that could be detected in lichens, new degenerate primers targeting ketoacylsynthase (KS) domains of specific clades of PKS genes have been developed and tested on various lichen samples. Using these primers, 19 KS domains from various lichens were obtained. Phylogenetic analysis of the KS domains was used to infer the function of the PKS genes based on the predicted PKS domain architecture and chemical analysis by TLC and/or HPLC. KS domains from PKS clades not previously known in lichens were identified; this included the clade III NR (non-reducing)-PKSs, PR (partially reducing)-PKSs and HR (highly reducing)-PKSs. The discovery of clade III NR-PKSs with C-methyltransferase (CMeT) domain and their wide occurrence in lichens was especially significant. Based on the KS domain phylogenetic analysis and compounds detected in the individual lichens, the clade III NR-PKSs were hypothesized to be involved in the biosynthesis of β-orsellinic acid and methylphloroacetopheno ne - the monoaromatic precursors for many lichen coupled phenolic compounds, such as β-orcinol depsides/depsidones and usnic acid. A strategy has been developed to isolate clade III NR-PKSs directly from environmental lichen DNA using clade III NR-type KS amplified from the degenerate primers (NR3KS-F/R) as homologous probes. Another pair of degenerate primers specific to the CMeT domain of NR-PKSs has also been developed to facilitate the cloning and probing of new clade III NR-PKS genes in lichens. A clade III NR-PKS gene (xsepks1) from X. semiviridis was cloned successfully. This is the first report of the isolation of a full-length PKS gene from environmental lichen DNA. The domain architecture of xsepks1 is KS-AT-ACP-CMeT, as expected for a clade III NR-PKS, suggesting that the newly developed clade-specific primers are useful for cloning new clade III NR-PKS genes and that KS domain phylogenetic analysis can predict the functional domains in PKSs. Attempts were made to characterize the function of xsepks1 by heterologous expression in Aspergillus species. Both A. nidulans (transformed with 5´partial xsepks1 including native promoter) and A. oryzae (transformed with full-length xsepks1 under the regulation of starch-inducible amyB promoter) were tested as potential hosts for the expression of lichen PKS genes. Transcriptional analysis showed that A. nidulans could potentially utilize the lichen PKS gene promoter and both fungal hosts could splice the introns of a lichen PKS gene. Several compounds unique to the A. oryzae transformants carrying xsepks1 were detected, but they could not be reproduced in subsequent fermentations even though the gene was transcribed into mRNA. None of the expected products (β-orsellinic acid, methylphloroacetophenone or similar methylated monoaromatic compounds) was detected in A. oryzae transformants, and the function of xsepks1 remains to be determined. The other clade III NR-PKS genes detected in X. semiviridis cou ld also be responsible for the biosynthesis of β-orsellinic acid or methylphloroacetophenone, as precursors of the major secondary metabolites detected in X. semiviridis (i.e. fumarprotocetraric acid, succinprotocetraric acid and usnic acid). Overall, the work in this thesis demonstrated the prospect of using a molecular approach to access the lichen biosynthetic potential without going through the cumbersome culturing stage.

Understanding how cellulose synthesis occurs is key to understanding the formation of the plant cell wall. This understanding could also be key to modifying cellulose production to permit more efficient extraction of glucose from cellulose for the production of biobased materials. Cellulose biosynthesis is carried out by cellulose synthases; transmembrane multimeric processive glycosyltransferases responsible for polymerizing UDP‐glucose into glucan chains. Thirty‐six glucan chains bind together in parallel to form elementary cellulose microfibrils. Due to the essential nature of cellulose synthases for plant survival and the recalcitrant nature of the cell wall to chemical and enzymatic digestion, the cellulose synthases can be very difficult to analyze by traditional approaches. In an attempt to circumvent some of the issues of studying cellulose synthases, the cellulose synthase genes CESA1 and CESA3, along with the cell wall associated genes COBRA, DET3 and POM1 were recombined into an engineered Autographa californica nucleopolyhedron virus and expressed in Spodoptera fruigiperda ovarian cells. Although recombinant protein could be detected for CESA1 and CESA3, C14‐glucose incorporation on baculovirus infected cell lines have given inconclusive results to the cellulose synthase activity of the CESA1 and CESA3 proteins. With further optimization of the protein expression of CESA1 and optimization of the variability in the C14‐glucose incorporation assays, the baculovirus system may prove a useful tool for studying the cellulose synthases. / UOIT

Calmodulin (CaM) is a calcium-binding protein that has promiscuous regulatory interactions with over three hundred intracellular protein targets. The focus of this study was to characterize the functional role of phosphorylated CaM in vitro and calcium-deficient CaM (Apo-CaM) ex vivo. In the in vitro study, the effect of phosphorylated CaM on the binding and activation of CaM target proteins was analyzed using mammalian Nitric Oxide Synthase (NOS). NOS is an enzyme that catalyzes the conversion of L-arginine to L-citrulline and •NO. In addition, the activation of NOS by modified CaM proteins was also analyzed in the presence of a CaM binding peptide, PEP-19. Protein trafficking experiments were performed ex vivo to extend our understanding of Apo-CaM’s functional role in mammalian cells. The cell lines that were used in this investigation include mouse Embryonic Stem Cells (mESC), Human Umbilical Vein Endothelia Cells (HUVEC) and Human Neuronal Glioma Cells (HNGC). The major finding of this projects are: phosphorylation of selective CaM residues can attenuated NOS activity, electrostatic interactions are important in the activation of iNOS by CaM, and the activation of iNOS by CaM occurs in a calcium-dependent manner

Calmodulin (CaM) is a calcium-binding protein that has promiscuous regulatory interactions with over three hundred intracellular protein targets. The focus of this study was to characterize the functional role of phosphorylated CaM in vitro and calcium-deficient CaM (Apo-CaM) ex vivo. In the in vitro study, the effect of phosphorylated CaM on the binding and activation of CaM target proteins was analyzed using mammalian Nitric Oxide Synthase (NOS). NOS is an enzyme that catalyzes the conversion of L-arginine to L-citrulline and •NO. In addition, the activation of NOS by modified CaM proteins was also analyzed in the presence of a CaM binding peptide, PEP-19. Protein trafficking experiments were performed ex vivo to extend our understanding of Apo-CaM’s functional role in mammalian cells. The cell lines that were used in this investigation include mouse Embryonic Stem Cells (mESC), Human Umbilical Vein Endothelia Cells (HUVEC) and Human Neuronal Glioma Cells (HNGC). The major finding of this projects are: phosphorylation of selective CaM residues can attenuated NOS activity, electrostatic interactions are important in the activation of iNOS by CaM, and the activation of iNOS by CaM occurs in a calcium-dependent manner

Thesis (M.S.)--Cleveland State University, 2008. / Abstracts. Title from PDF t.p. (viewed on Jan. 29, 2009). Includes bibliographical references (p. 33-36). Available online via the OhioLINK ETD Center. Also available in print.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xii, 152 p.; also includes graphics (some col.). Includes bibliographical references (p. 137-150). Available online via OhioLINK's ETD Center