This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

Malaria is a disease of major public health importance, killing approximately one million people and causing about 250 million cases of fever annually. It mostly affects children under the age of five and pregnant women in many developing countries, making it a prominent issue in international health and maternal and child health. The most aggressive form of malaria is caused by the parasite Plasmodium falciparum which is responsible for 80% of infections and 90% of deaths from malaria, and is most prevalent in sub-Saharan Africa. Public Health interventions include the implementation of prevention programs, health education, and chemotherapy. The latter has experienced multiple problems in the past years whereby resistance of the parasite to the available drugs has emerged, rendering the majority of them ineffective. Furthermore, the high cost of those drugs represents a major obstacle to their dispensation in areas of the world where the affected people are often the less fortunate. The enzyme Cytochrome b5 Reductase (cb5r) and its electron acceptor Cytochrome b5 (cb5) play a role in fatty acid elongation, cholesterol biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. Therefore, these proteins are suitable as potential novel drug targets for malaria. These two proteins have been thoroughly studied in mammals but have to be characterized in microorganisms such as fungi and parasites, including Plasmodium falciparum. It is important to note that plant cb5r has been identified as a novel herbicidal target. Considering the close phylogenetic relationship between plant cb5r and Plasmodium falciparum cb5r, we conclude that these plant inhibitors may also serve as promising candidates for a new class of antimalarial drugs against the parasite. In this project, we want to obtain the biochemical and enzymatic characterization of cb5r and cb5 in order to establish whether these two proteins represent potential novel drug targets in Plasmodium falciparum malaria. This initial work may lead to the development of novel drugs which will consequently affect the field of public health with respect to drug delivery, drug resistance, and drug chemotherapy.

Försäljare av detoxpreparat hävdar att kroppens reningsmekanismer i vårt moderna samhälle inte längre räcker till och behöver hjälp på vägen, företrädesvis i form av påstått utrensande örtblandningar. Vissa förespråkar även tarmsköljningar och joniserande fotbad. Syftet med den här studien är att kartlägga floran av detoxprodukter i form av kosttillskott: vilka typer det finns, vilka ingredienser som ingår och vilka påståenden som görs. Påståendena har klassificerats efter testbarhet och trovärdigheten har bedömts genom sökningar i PubMed och Web of Science. Några av de testbara påståendena verkar helt sakna evidens, något påstående lutar sig mot studier behäftade med problematiska brister medan vissa påståenden förtjänar vidare studier, då de bygger på studier med preliminärt lovande resultat. Det går inte att utesluta en framtid för evidensbaserade detoxdieter, lika lite som det går att säga att detoxdieter av idag håller vad de lovar. Det krävs fler kliniska, placebokontrollerade studier och en tydligare skiljelinje mellan det som är ren bluff och det som är seriös forskning. Konsumenter bör vara medvetna om bristen på evidens och riskerna som finns, exempelvis i form av kontaminerade produkter och risk för interaktioner med läkemedel. / Sellers of detox preparations argue that in our modern society, the body's purification mechanisms are no longer sufficient and need a little help, preferably in the form of alleged cleansing herbal formulas. Some also advocate colonic irrigation and ionizing foot baths. The purpose of this study is to survey the flora of detox products in the form of nutritional supplements: what types there are, what ingredients are included and which claims are made. The claims have been classified for testability and reliability was assessed through searches in PubMed and Web of Science. Some of the testable assertions seem to completely lack evidence, other claims lean on studies that suffer from problematic deficiencies, while some claims deserve further study since they find support from studies with promising preliminary results. You cannot rule out a future for evidence-based detox diets, no more than you can to say that detox diets today keep their promises. More clinical, placebo-controlled studies are required and a clearer dividing line between what is pure bluff and what is serious research. Consumers should be aware of the lack of evidence and the risks that exist, for example in the form of contaminated products and the potential risk of interactions with medicines.

No description available.

Humans are continuously challenged by harmful endogenous and xenobiotic substances. Detoxification is the ability to neutralise and remove these substances from the body. Glycine N-acyltransferase, EC 2.3.1.13 (GLYAT) is a key enzyme in detoxification. GLYAT catalyses an amino acid (glycine) conjugation reaction in phase II of detoxification. It is expected that, similar to what has been observed in the Cytochrome P450 enzymes, variations within the GLYAT gene may lead to altered enzyme activity that may affect the efficacy of detoxification. The aim of this study was to identify genetic variations within the GLYAT gene of a cohort of individuals whose GLYAT activity has been biochemically characterized. Biochemical profiles of phase I and II detoxification of a number of individuals was screened to select those with possible aberrant GLYAT activity. Eighteen selected individuals agreed to participate in the study. The 23.21 kb GLYAT gene of the participants was amplified in four fragments and sent for pyrosequencing (Roche GS FLX titanium) at Inqaba Biotec. The results were analysed with the Lasergene software package from DNAStar (Madison, Wisconsin, USA). A total of 94 variations were identified from the Next Generation Sequencing data. Of these three found in the exons were known variations and four variations located in the exons were novel. A total of 62 known and 25 novel variations were identified in the introns of the GLYAT gene. Sanger sequencing verified 70.29% (68 in total) of the variation, which included 12 novel variations, of which one is located in exon six. Real-time quantitative PCR (qPCR) experiments were conducted and the data analysed using CopyCaller software to identify copy number variations within the cohort. It was found that participant 17 may have multiple copies of parts of the 3-terminal end of the gene (exons five and six), which might have an effect on GLYAT activity. Variations could possibly affect GLYAT activity, but the data was inconclusive and must be confirmed. Some of the variations could possibly affect GLYAT activity, but no correlation could be made between the variations identified during this study and the cohort’s detoxification ability. Further studies needs to be conducted to establish the effect of the variations in combination with one another on GLYAT activity. If some of these variations affect GLYAT activity such data might shed some light on variations observed between the glycine conjugation ability of individuals. Such information could eventually be of value in treatment of inborn errors of metabolism. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Leishmania are protozoan parasites that are transmitted by a sand fly vector. These parasites affect not only humans but also wild animals including domestic dogs and rodents, which form an additional challenge and public health problem to control the disease. Leishmaniasis is an important disease with worldwide distribution, including Saudi Arabia, the Middle East, and other tropical and subtropical areas around the world. Due to the expansion of irrigation and agricultural activities, more exposure to sand fly occurs, which leads to the expansion of leishmaniasis infections as newly emerging disease. Emerging drug resistance in leishmaniasis is an additional problem, contributed by enzymes involved in the detoxification of pharmacological agents and other xenobiotics. Cytochrome b5 reductase (Cb5r) has a high pharmacological significance because of its essential role in fatty acid elongation, biosynthesis of cholesterol (humans) or ergosterol (Leishmania, fungi), and cytochrome P450-mediated detoxification of xenobiotics. Leishmania Cb5r has seven different isoforms whereas human has only one. Cb5r-7 isoform in Leishmania has closest homology to the human Cb5r. The three specific aims of this thesis project are focusing on (i) cloning of the Cb5r-7 isoform from Leishmania mexicana, (ii) its purification as recombinant His-tagged protein from E.coli, and (iii) its functional characterization as potential pharmacological target against Leishmania.

Šio darbo tikslas: Nustatyti, įvertinti ir palyginti ekologiškoje ir įprastinėje grūdinėje žaliavoje mikologinį ir mikotoksikologinį užterštumą, panaudojant pienarūgštes bakterijas deoksinivalenolio detoksikacijai. Darbo uždaviniai: Ištirti, įvertinti ir palyginti ekologiniuose ir įpratiniuose ūkiuose surinktų grūdų mikologinį ir mikotoksikologinį užterštumą. Įvertinti ir palyginti kvietinių ir ruginių miltų mikotoksikologinį užretštumą. Įvertinti fermentacinių procesų įtaką DON kiekiui duonos gamybos grandinėje Naudota metodika: Mikromicetų skaičius įvertintas pagal LST ISO 6611:2004. Mikromicetų rūšys identifikuotos vadovaujantis apibūdintojais (Samson et al., 2000; Lugauskas ir kt., 2002). Mikotoksinų koncentracija nustatyta imunofermentinės analizės metodu (IFA), panaudojus komercinius VERATOX® DON 5/5, (Neogen, JAV) rinkinį. Fermentacija atlikta panaudojus pieno rūgšties bakterijų kultūrą L. sakei. Tyrimų rezultatai pateikti panaudojus R statistinį paketą. Išvados: Ištyrūs kviečius ir rugius surinktus iš ekologinių ir iprastinių ūkių daugiausia dominavo Fusarium spp. genties mikromicetai. Potencialūs deoksinivalenolio producentai. Gyvybingų sporų skaičius 33 proc. didesnis nustatytas kviečiuose surinktuose iš ekologinių ūkių (p<0,05). Lyginant rugių mėginius, gyvybingų sporų skaičius 29,5 proc. didesnis nustatytas taip pat ekologiniuose ūkiuose surinktuose rugiuose 44±2,1x102 KSV/g (p<0,05). Palyginus gyvybingų sporų skaičių kvietiniuose ir ruginiuose miltuose 5... [toliau žr. visą tekstą] / Objective of the thesis: To identify, evaluate and compare mycological and mycotoxicological contamination in ecological and standard grains by using lactic acid bacteria for deoxynivalenol detoxification. Tasks: Investigate, evaluate and compare mycological and mycotoxicological contamination of grains collected in ecological and standard farms. Evaluate and compare mycotoxicological contamination of wheat and rye flour. Evaluate influence of fermentation processes on DON level in bread production chain. Research methodology: The total count of microorganisms (CFU/g-1) in the analysed sample was determined by way of dilution using the nutrient agar. LST ISO 6611:2004. Micromycete content was determined according to the method of enzyme-linked immunosorbent assay (ELISA) by using the commercial VERATOX® DON 5/5, (Neogen, JAV). Fermentation processes using latic acid bacteria L. sakei. The results of the data analysis and research presented using the R statistical package. Results and discussion: After examining wheat and rye that were collected from ecological and standard farms, it was noticed that mycromycetes of Fusarium spp. genus dominated mostly. Potential Deoxynivalenol Prudecents. Wheat that was collected from ecological farms had 33 proc. higher number of viable spores, i.e. (p<0,05). Similarly, the number of viable spores of rye was 29.5 proc. higher in the rye that was collected in the ecological farms, i.e. 44±2,1x102 CFU/g (p<0,05). (CFU-Colony forming... [to full text]

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.

Diss. (sammanfattning) Karlstad : Karlstads universitet, 2009. / Härtill 5 uppsatser.

Pfaffia glomerata (Spreng) Pedersen, also known as Brazilian ginseng, has been used in popular medicine due to its important medicinal qualities. This study aims to characterize the nutrient needs of P. glomerata, (Chapter 1) as well as to assess the tolerance of this plant to high levels of cadmium in the substrate (Chapter 2). P. glomerata plants produced in vitro and acclimatized ex vitro were used as initial material. In Chapter 1, three (3) experiments are described. In the first experiment, under greenhouse conditions, young plants of P. glomerata were cultivated in Paleudalf soil with different treatments for fertilization: control (without fertilization), complete fertilization (with macro and micronutrients) and the individual omissions of N, P, K, Ca, Mg, S and micronutrients (Zn, B, Cu, Fe, Mn and Mo). In the second experiment, in greenhouse, the plants were submitted to twenty-seven (27) treatments of fertilization in complete trifactorial system (3x3x3), represented by a combination of three levels (40, 80 e 120 mg kg-1) of N, P and K in the fertilization, using the same soil as substrate. In the third experiment, under open field conditions, six (6) treatments involving the combination of two levels (50 e 100 kg ha-1) of N, P and K in the fertilization in two periods of cultivation, sixteen (16) and six (6) months, were evaluated respectively. It was observed in the fertilization in the experiment with the missing nutrient technique that the young plants of P. glomerata showed great decrease in biomass growth as well as in the macro and micronutrients by the omission of the nutrients N, K, S and P. In the second experiment, in greenhouse, the production of biomass by different organs of the plant responded differently to the triple interaction of NPK fertilizations, in which the dosages of maximum technical efficiency estimated (DMET) to the fertilizations of N, P and K in the production of total biomass of the plant were 60, 120 and 80 mg kg-1 respectively. In the open field, the NPK fertilization affected the production of biomass of the organs of the plants in different ways in 16 months of cultivation. In order to have a greater accumulation of mineral nutrients in the tissues of P. glomerata and a possible relation to the accumulation of active principles of medicinal interests, the plants should be collected after six (6) months of field cultivation. In the study described in Chapter 2, an experiment with seedlings of Pfaffia glomerata cultivated under the hydroponic system with five levels of cadmium (Cd) (0, 20, 40, 60 e 80 μm) was carried out during seven days. Those plants demonstrated an increase in the content of Cd in the roots and shoots. The total biomass was reduced significantly in 80 μM of Cd. The activity of the ALA-D (delta-aminolevulinic acid dehydratase) and AP (acid phosphatase) was reduced with the levels of Cd. The reduction in the activity of ALA-D was accompanied by the content of chlorophyll. Enzymatic antioxidants, such as catalase and ascorbate peroxidase, as well as non-enzymatic antioxidants like ascorbic acid and non-protein thiol groups had an important role in the detoxification of effects induced by Cd in the roots and shoot of the seedlings of P. glomerata. / A Pfaffia glomerata (Spreng) Pedersen, conhecida como ginseng brasileiro, tem sido utilizada na medicina popular em função de suas importantes qualidades medicinais. O estudo teve por objetivo caracterizar as necessidades nutricionais da P. glomerata (Capítulo 1), bem como avaliar a tolerância dessa planta a altos níveis de cádmio no substrato (Capítulo 2). Plantas de P. glomerata produzidas in vitro e aclimatizadas ex vitro foram usadas como material inicial. No estudo referente ao Capítulo 1, três experimentos foram realizados. No primeiro experimento, sob condições de casa de vegetação, plantas jovens de P. glomerata foram cultivadas em Argissolo Vermelho distrófico arênico com diferentes tratamentos de adubação: controle (sem adubação), adubação completa (com macro e micronutrientes) e as omissões individuais de N, P, K, Ca, Mg, S e dos micronutrientes (Zn, B, Cu, Fe, Mn e Mo). No segundo experimento, ainda em casa de vegetação, plantas foram submetidas a 27 tratamentos de adubação, em esquema trifatorial completo (3x3x3), representados pela combinação de três níveis (40, 80 e 120 mg kg-1) de N, de P e de K na adubação, utilizando o mesmo solo como substrato. No terceiro experimento, sob condições de cultivo a campo foram avaliados seis tratamentos constituídos da combinação de dois níveis (50 e 100 kg ha-1) de N, de P e de K na adubação, em duas épocas de cultivo, 16 e 6 meses, respectivamente. No experimento com a técnica do nutriente faltante, verificou-se que plantas jovens de P. glomerata apresentaram maior redução em biomassa, bem como nos teores de macro e micronutrientes, pela omissão de N, K, S e P. No segundo experimento, ainda em casa de vegetação, a produção de biomassa pelos diferentes órgãos da planta respondeu distintamente à interação tripla das adubações NPK, onde as doses de máxima eficiência técnica estimada com as adubações de N, P e K e a produção de biomassa total da planta, foram de, respectivamente, 60, 120 e 80 mg kg-1. No cultivo a campo, a adubação NPK afetou distintamente a produção de biomassa dos órgãos das plantas com 16 meses de cultivo. Para uma maior acumulação de nutrientes minerais nos tecidos de P. glomerata e uma possível relação com o acúmulo de princípios ativos de interesse medicinal, as plantas devem ser coletadas após os 6 meses de cultivo a campo. No estudo referente ao Capítulo 2, um experimento foi realizado com plântulas de Pfaffia glomerata cultivadas sob sistema hidropônico, com cinco níveis de cádmio (Cd) (0, 20, 40, 60 e 80 μM), durante 7 dias. Essas plantas apresentaram aumento no conteúdo de Cd nas raízes e parte aérea. A biomassa total foi apenas reduzida significativamente a 80μM de Cd. A atividade da ALA-D (Ácido delta aminolevulínico desidratase) e da AP (fosfatase ácida) foram reduzidas com os níveis de Cd. A redução na atividade da ALA-D foi acompanhada pelo conteúdo de clorofila. Os antioxidantes enzimáticos, como a catalase e a ascorbato peroxidase, bem como antioxidantes não enzimáticos, como o ácido ascórbico e os grupos tióis não protéicos, tiveram um papel chave na destoxificação de efeitos induzidos pelo Cd nas raízes e parte aérea das plântulas de P. glomerata.