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Gilvocarcin V (GV) belongs to the angucycline class of antibiotics that possesses remarkable anticancer and antibacterial activities with low toxicity. Gilvocarcin exhibits its light induced anticancer activity by mediating crosslinking between DNA and histone H3. When photo-activated by near-UV light, the C8 vinyl group forms a [2+2] cycloadduct with thymine residues of double stranded DNA. D-fucofuranose is considered essential for histone H3 interactions. However, the poor water solubility has rendered it difficult to develop gilvocarcin as a drug. We aim to design novel gilvocarcin analogues with improved pharmaceutical properties through chemo-enzymatic synthesis and mutasynthesis. Previous studies have characterized many biosynthetic genes encoding the gilvocarcin biosynthetic skeleton. Despite these previous findings the exact functions of many other key genes are yet to be fully understood. Prior gene inactivation and cross-feeding experiments have revealed that the first isolable tetracyclic aromatic product undergoes a series of steps involving C–C bond cleavage followed by two O-methylations, a penultimate C-glycosylation and final lactone formation in order to fully develop the gilvocarcin structure. To provide a deeper understanding of these complex biochemical transformations, three specific aims were devised: 1) synthesis of the proposed intermediate and in vitro enzyme reactions revealed GilMT and GilM’s roles in gilvocaric biosynthesis; 2) utilizing in vitro studies the enzyme responsible for the C–C bond cleavage and its substrate were determined; 3) a small series of structural analogues of the intermediate from the gilvocarcin pathway was generated via chemical synthesis and fed to the mixture of the enzymes, GilMT and GilM. These reaction mixtures were then analyzed to establish the diversity of substrates tolerated by the enzymes.

A recent epidemiological study suggested that higher caffeine intake reduces the risk of Alzheimer's disease (AD). Caffeine, a widely consumed stimulatory drug, is a non-selective adenosine receptor antagonist that has been shown to increase plasma adenosine levels in rodents. To determine any long-term protective effects of caffeine in a controlled longitudinal study, caffeine was added to the drinking water of APPsw transgenic (Tg) mice between 4 and 9 1/2 months of age, with behavioral testing done during the last 6 weeks of treatment. The average daily intake of caffeine per mouse (1.5 mg) was the human equivalent of 5 cups of coffee/day. Across multiple cognitive tasks of spatial learning/reference memory, working memory, and recognition/identification, Tg mice given caffeine (Tg+Caff) performed significantly better than Tg control mice and similar to non-transgenic controls. Discriminant Function Analysis involving multiple cognitive measures clearly showed the superior overall cognitive performance of Tg+Caff mice compared to Tg controls. Analysis of Aβ in the hippocampus by ELISA revealed Tg+Caff mice had significantly less soluble Aβ1-40 and insoluble Aβ1-42. In a follow-up study involving neurochemical analysis only, caffeine was added to the drinking water of 17 month old APPsw mice for 18 days. In this study, Tg+Caff mice also showed a significant reduction of insoluble Aβ1-42 in the hippocampus. In contrast to the reduced extracellular brain levels of adenosine in Tg controls, caffeine treatment normalized brain adenosine levels in Tg mice to that of non-transgenic controls. Analysis of amyloidogenic secretase activity revealed the reduction in Αβ is likely because of a reduction in gamma secretase activity as a result of increased SAM silencing of PS1 expression. This study suggest that a modest, long-term caffeine intake of approximately 500 mg per day (5 cups of coffee) may reduce considerably the risk of AD by decreasing amyloidogenesis.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [73]-84).

Thesis (M.S.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 57 pages. Includes Vita. Includes bibliographical references.

A contributing factor in recurrent Listeria monocytogenes (L. monocytogenes) food contamination is that this bacterium produces biofilms on surfaces to persist in food-processing environments. Quorum sensing (QS) is a cell-to-cell communication system utilized by bacteria within biofilms to collaborate and adapt to environmental stresses. However, the details of how the QS-dependent network contributes to biofilm development of L. monocytogenes have yet to be well understood. By comparing the transfer rates of planktonic and biofilm (sessile) L. monocytogenes from stainless steel blades to bologna slices, we found that sessile bacteria had reduced transferability onto a single slice but caused the increase in the number of contaminated slices. This suggests that physiological adaptions derived during biofilm development affect bacterial dissemination. Given the contribution of proteins and environmental temperatures to the extracellular polymeric substances (EPS) synthesis and biofilm integrity, we evaluated the exoproteomes of biofilms formed at 25 and 37°C using 2D-gel electrophoresis and LC-MS/MS. We found exoproteases Lmo0186, Cwh, and Spl exclusively in biofilms formed at 25°C and their greater expression in the gene level at 25°C. By using the zymography and crystal-violet-staining assay with a protease inhibitor, we observed a greater proteolytic activity at lower temperatures and showed that the attenuated proteolytic activity of proteases is positively correlated with increased biofilmorming ability at 25°C. Considering the transcriptional role of QS systems during biofilm development, we investigated how the accessory gene regulator (Agr)-based and metabolite S-Adenosylmethionine (SAM)-involved QS systems modulate nutrient availability and EPS synthesis. The results revealed that the SAM signal interacts with the Agr QS at the transcriptional level during biofilm development, whereas SAM and Agr QS regulate distinct EPS synthesis pathways. Additionally, this interaction is dependent on bacterial life modes (planktonic and sessile). Overall, we conclude that L. monocytogenes manipulates the synthesis of EPS with the coregulation of metabolism and QS for biofilm formation and the production of exoproteases for biofilm dispersion. These precise regulations on EPS enable L. monocytogenes to prolong its survival and promote its dissemination in environments.

O estado de metilação é suscetível a mudanças quando os organismos são expostos a agentes ambientais tais como componentes dos alimentos e medicamentos. Uma dieta rica em metionina (Met) poderia modular a concentração de S-adenosilmetionina (SAM) e S-adenosilhomocisteína (SAH) e alterar o estado de metilação da região promotora de genes supressores de tumores. Tanto a hipometilação global quanto a hipermetilação de genes específicos estão envolvidas na instabilidade genômica e poderiam resultar em dano ao DNA. Este estudo avalia se a dieta suplementada com Met associada a doxorrubicina (DXR), um fármaco antitumoral que induz espécies reativas, resulta em alterações no estado de metilação da região promotora do gene TP53, na razão SAM/SAH, na concentração de glutationa (GSH) e em dano ao DNA. Quarenta ratos machos Wistar foram separados em dois grupos: dieta suplementada com Met (ração comercial acrescida de 2% Met) e dieta controle (ração comercial) por seis semanas. Cada grupo foi subdividido em dois subgrupos que receberam DXR (1mg/Kg) ou solução salina intraperitoneal na terceira e sexta semanas de tratamento. Os rins e fígado foram utilizados para isolamento do DNA, determinação da concentração de SAM, SAH e GSH, e análise da instabilidade genômica. Todos os grupos apresentaram o mesmo estado de metilação da região promotora do gene TP53, determinado pelo método de análise de restrição combinada com bissulfito (COBRA). Este fato poderia ser explicado pelo índice de metilação (razão SAM/SAH) que permaneceu inalterado, possivelmente devido a uma adaptação do ciclo da Met que manteve a concentração de SAM. A depleção de GSH não ocorreu quando DXR foi associada a dieta suplementada com Met. Portanto, a suplementação com Met manteve a concentração de GSH em ratos tratados com DXR. A dieta suplementada com Met não induziu instabilidade genômica e não alterou o dano ao DNA induzido pela DXR. Em conclusão, DXR induz depleção de GSH que é inibida pela suplementação com Met. Entretanto, a mesma suplementação não previne a instabilidade genômica induzida pela DXR. A dieta suplementada com Met aumenta a concentração de SAH renal sem alterar a concentração de SAM e GSH. Tanto a dieta suplementada quanto a DXR não induzem hipermetilação na região promotora do gene TP53. / The DNA methylation status is susceptible to changes when organisms are exposed to environmental agents such as food components and drugs. A methionine-rich (Met) diet may modulate S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations, which could change the DNA methylation status in the promoter region of tumor suppressor genes. Global hipomethylation and gene-specific hipermethylation are involved in genomic instability and it could result in DNA damage. This study intends to evaluate if a Met-rich diet associated with doxorubicin (DXR), an antitumoral drug that induces reactive species, result in changes in the methylation status of the TP53 gene promoter, in the SAM/SAH ratio, in glutathione levels (GSH) and in DNA damage. Forty male Wistar rats were separated into two groups: Met-rich diet (standard chow plus 2% Met), and control diet (standard chow) for six weeks. Each group was subdivided into another two groups that received DXR (1mg/kg) or saline intraperitoneally in the third and sixth weeks of the experiment. The kidneys and the liver were removed for DNA isolation, SAM, SAH and GSH determination, and genomic instability assay. All groups showed the same unmethylated status in the TP53 promoter according to the Combined Bisulfite Restriction Analysis (COBRA). This could be explained by the fact that the methylation index (SAM/SAH ratio) remained unchanged, possibly because of an adaptive Met pathway that maintains SAM levels. GSH depletion did not occur when DXR was associated with the Met-rich diet. As a matter of fact, the Met-rich diet improved GSH concentration in DXR-treated rats. Met-rich diet did not induce genomic instability, and it did not alter DNA damage induced by DXR. In conclusion, DXR induces GSH depletion which is inhibited by Met supplementation. However, Met-rich diet may not prevent genomic instability induced by DXR. A Met-rich diet increases SAH levels; however, it does not change GSH and SAM levels. Neither Met supplementation nor DXR induced DNA hypermethylation in the TP53 gene promoter.

O estado de metilação é suscetível a mudanças quando os organismos são expostos a agentes ambientais tais como componentes dos alimentos e medicamentos. Uma dieta rica em metionina (Met) poderia modular a concentração de S-adenosilmetionina (SAM) e S-adenosilhomocisteína (SAH) e alterar o estado de metilação da região promotora de genes supressores de tumores. Tanto a hipometilação global quanto a hipermetilação de genes específicos estão envolvidas na instabilidade genômica e poderiam resultar em dano ao DNA. Este estudo avalia se a dieta suplementada com Met associada a doxorrubicina (DXR), um fármaco antitumoral que induz espécies reativas, resulta em alterações no estado de metilação da região promotora do gene TP53, na razão SAM/SAH, na concentração de glutationa (GSH) e em dano ao DNA. Quarenta ratos machos Wistar foram separados em dois grupos: dieta suplementada com Met (ração comercial acrescida de 2% Met) e dieta controle (ração comercial) por seis semanas. Cada grupo foi subdividido em dois subgrupos que receberam DXR (1mg/Kg) ou solução salina intraperitoneal na terceira e sexta semanas de tratamento. Os rins e fígado foram utilizados para isolamento do DNA, determinação da concentração de SAM, SAH e GSH, e análise da instabilidade genômica. Todos os grupos apresentaram o mesmo estado de metilação da região promotora do gene TP53, determinado pelo método de análise de restrição combinada com bissulfito (COBRA). Este fato poderia ser explicado pelo índice de metilação (razão SAM/SAH) que permaneceu inalterado, possivelmente devido a uma adaptação do ciclo da Met que manteve a concentração de SAM. A depleção de GSH não ocorreu quando DXR foi associada a dieta suplementada com Met. Portanto, a suplementação com Met manteve a concentração de GSH em ratos tratados com DXR. A dieta suplementada com Met não induziu instabilidade genômica e não alterou o dano ao DNA induzido pela DXR. Em conclusão, DXR induz depleção de GSH que é inibida pela suplementação com Met. Entretanto, a mesma suplementação não previne a instabilidade genômica induzida pela DXR. A dieta suplementada com Met aumenta a concentração de SAH renal sem alterar a concentração de SAM e GSH. Tanto a dieta suplementada quanto a DXR não induzem hipermetilação na região promotora do gene TP53. / The DNA methylation status is susceptible to changes when organisms are exposed to environmental agents such as food components and drugs. A methionine-rich (Met) diet may modulate S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations, which could change the DNA methylation status in the promoter region of tumor suppressor genes. Global hipomethylation and gene-specific hipermethylation are involved in genomic instability and it could result in DNA damage. This study intends to evaluate if a Met-rich diet associated with doxorubicin (DXR), an antitumoral drug that induces reactive species, result in changes in the methylation status of the TP53 gene promoter, in the SAM/SAH ratio, in glutathione levels (GSH) and in DNA damage. Forty male Wistar rats were separated into two groups: Met-rich diet (standard chow plus 2% Met), and control diet (standard chow) for six weeks. Each group was subdivided into another two groups that received DXR (1mg/kg) or saline intraperitoneally in the third and sixth weeks of the experiment. The kidneys and the liver were removed for DNA isolation, SAM, SAH and GSH determination, and genomic instability assay. All groups showed the same unmethylated status in the TP53 promoter according to the Combined Bisulfite Restriction Analysis (COBRA). This could be explained by the fact that the methylation index (SAM/SAH ratio) remained unchanged, possibly because of an adaptive Met pathway that maintains SAM levels. GSH depletion did not occur when DXR was associated with the Met-rich diet. As a matter of fact, the Met-rich diet improved GSH concentration in DXR-treated rats. Met-rich diet did not induce genomic instability, and it did not alter DNA damage induced by DXR. In conclusion, DXR induces GSH depletion which is inhibited by Met supplementation. However, Met-rich diet may not prevent genomic instability induced by DXR. A Met-rich diet increases SAH levels; however, it does not change GSH and SAM levels. Neither Met supplementation nor DXR induced DNA hypermethylation in the TP53 gene promoter.

Malaria is prevalent in over 100 countries which is populated by half of the world’s population and culminates in approximately one million deaths per annum, 85% of which occurs in sub-Saharan Africa. The combined resistance of the mosquitoes and parasites to the currently available pesticides and antimalarial chemotherapeutic agents requires the concerted effort of scientists in the malaria field to identify and develop novel mechanisms to curb this deadly disease. In this study, a thorough understanding of the role players in the polyamine pathway of the parasite was obtained, which could aid future studies in the development of novel inhibitory compounds against these validated drug targets. The uniquely bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) of Plasmodium falciparum forms an important controlling node between the polyamine and methionine metabolic pathways. It has been speculated that the unique bifunctional association of the rate-limiting enzymes allows for the concerted regulation of the respective enzyme activities resulting in polyamine synthesis as per requirement for the rapidly proliferating parasite while the methionine levels are strictly controlled for their role in the methylation status. The results of this study showed that the enzyme activities of the bifunctional complex are indeed coordinated and subtle conformational changes induced by complex formation is suggested to result in these altered kinetics of the individual AdoMetDC and ODC domains. Studies also showed that the identification of the interaction sites between the domains, which allows for communication across the complex, may be targeted for specific interference with the enzyme activities. Furthermore, these studies showed that the current knowledge on the different subclasses of the AdoMetDC family should be re-evaluated since P. falciparum AdoMetDC shows diverse properties from orthologues and therefore points towards a novel grouping of the plasmodial protein. The extensive biochemical and biophysical studies on AdoMetDC has also provided important avenues for the crystallisation and solving of this protein’s 3D structure for subsequent structure-based identification of drug-like lead compounds against AdoMetDC activity. The application of structure-based drug design on malarial proteins was additionally investigated and consequently proved that the rational design of lead inhibitory compounds can provide important scaffold structures for the identification of the key aspects that are required for the successful inhibition of a specific drug target. Spermidine synthase, with its intricate catalytic mechanism involving two substrate binding sites for the products of the reactions catalysed by AdoMetDC/ODC, was used to computationally identify compounds that could bind within its active site. Subsequent testing of the compounds identified with a dynamic receptor-based pharmacophore model showed promising inhibitory results on both recombinant protein and in vitro parasite levels. The confirmation of the predicted interaction sites and identification of aspects to improve inhibitor interaction was subsequently investigated at atomic resolution with X-ray protein crystallography. The outcome of this doctoral study shows the benefit in applying a multidisciplinary and multinational approach for studying drug targets within the malaria parasite, which has led to a thorough understanding of the targets on both biochemical and structural levels for future drug design studies. / Thesis (PhD)--University of Pretoria, 2011. / Biochemistry / unrestricted

Cysteinyl peptides likely played an important role in the prebiotic synthesis of cofactors, such as iron-sulfur clusters. However, cysteinyl peptides must be reduced in order to coordinate iron-sulfur clusters. Mixtures of ferric ions and cysteinyl peptides leads to the reduction of ferric to ferrous ions and the concomitant formation of disulfide bridged, oxidized cysteinyl peptides that are incapable of coordinating an iron-sulfur cluster. Here, we develop a photochemically driven solution to this problem. Lipoic acid (( R )-5-(1,2-dithiolane-3-yl)pentanoic acid), a molecule structurally similar to fatty acids, can be photochemically reduced and can subsequently reduce the oxidized cysteinyl peptides needed for the coordination of an iron-sulfur cluster. Other dithilane ring containing molecules possess similar activity to lipoic acid. The synthesis of small peptides containing cysteines, such as glutathione and GCG (Gly-Cys-Gly) is easy by both solid phase and solution phase methodologies. However, as the length of the peptide increases, the yield begins to decrease, especially for peptides containing cysteines due to oxidation. One solution could be to exploit a previously uncovered mechanism for the joining of peptides into longer peptides. Such mechanisms, referred to as CPL for catalytic peptide ligation, rely on either thiols or metals as catalysts and peptide nitriles as substrates. Thus far, CPL has only been exploited with non-cysteinyl peptides. In this thesis, we extend CPL to cysteine containing peptides by taking advantage of the templating effects of Zn2+. Longer peptides with properly spaced cysteines are frequently better able to stabilize iron-sulfur clusters in aqueous solution than shorter peptides. Coordination can either be complete or an open coordination position, filled by solvent, can be used to bind substrate. Two well-known examples of such an arrangement are the radical SAM (S-adenosylmethionine) enzyme and aconitase being an enzyme of the citric acid cycle. We designed and synthesized peptide sequences that could coordinate a [4Fe-4S]2+ cluster with three cysteinyl ligands, leaving an open coordination position. The stability of the [4Fe-4S] cluster was affected by the intermediates of the citric acid cycle. The iron-sulfur can be reconstituted with the long peptidyl sequences from proteins such as SLC25A39 which contains four cysteine ligands to form [2Fe-2S] cluster, which is necessary for glutathione transport from cytosol to mitochondria.