京都大学 / 新制・課程博士 / 博士(人間・環境学) / 甲第23272号 / 人博第987号 / 新制||人||233(附属図書館) / 2020||人博||987(吉田南総合図書館) / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 船曳 康子, 教授 久代 恵介, 教授 森谷 敏夫 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of Pf Pdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified Pf Pdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in Pf Pdx1, possibly allowing the molecule to enter the R5P-binding site of Pf Pdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting Pf Pdx1 was investigated using transgenic cell lines over-expressing Pf Pdx1 and Pf Pdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of Pf Pdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that Pf Pdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Biochemistry / Unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18599号 / 農博第2086号 / 新制||農||1027(附属図書館) / 学位論文||H26||N4874(農学部図書室) / 31499 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 植田 充美, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The vitamin B-6 content of three foods, roast top round of beef in au jus, canned green beans in pot liquor, and whole baked potatoes was measured. These foods are typically served together as choices on the six week cycle menu at three dining halls on the University campus. Random samples were taken when the foods were raw, just cooked, and held for one, two, and three hours. Two additional samples of roast beef were selected as the beef received more cooking and holding treatments. Saccharomyces uvarum was the test organism used in the A.O.A.C. microbiological analyses for vitamin B-6 quantitation. Total vitamin B-6 was measured, not the individual vitamers. Roast beef lost total vitamin B-6, sometimes in significant quantities at every sample time. Green beans followed the same pattern of loss with cooking and increased holding times. Some of the vitamin was leached into the au jus and pot liquor as they were held with the beef and beans, respectively. Whole baked potatoes also lost in vitamin B-6 content with each sample time; however, this loss was not always significant. Institutional cooking and holding procedures utilized for these selected foods had adverse effects on their vitamin B-6 content. / M.S.

A high performance liquid chromatographic (HPLC) system consisting of a binary mobile phase, a µBondapak C₁₈ column, and a fluorescence detector was used to separate the B-6 vitamers pyridoxal (PL), pyridoxine CPN), and pyridoxamine (PM) as well as the internal standard deoxypyridoxine. An extraction procedure, compatible with the HPLC system, was developed to isolate the B-6 vitamers from rat livers, kidneys, and brains. PL values were derived from the sum of tissue PL and pyridoxal phosphate (PLP), the latter having been dephosphorylated to PL. Similarly, PN values were the sum of tissue PN and pyridoxine phosphate (PNP) and PM data the sum of PM and pyridoxamine phosphate (PMP). Separation and quantitation of tissue vitamers were successful and reflected significant relationships between rat vitamin B-6 intakes and tissue concentrations of the B-6 vitamers PL and PM as well as total B-6 vitamer concentration. PM was found to be the major vitamer in all tissues. The HPLC data were compared to values obtained by the AOAC microbiological (MB) assay performed on the same samples. Significant correlations (r) ranging from 0.31 - 0.73 for PL, 0.30 - 0.47 for PM, and 0.48 - 0.55 for total B-6 vitamer concentration were observed between values obtained via the methods. Both HPLC and MB vitamer data approximated reported values. The HPLC method offered the advantages of relative speed and simplicity. / M.S.

The vitamin B-6 status of seemingly healthy adolescent girls was determined using several accepted and proposed parameters in an effort to establish guidelines for status evaluation. HPLC-derived plasma B-6 vitamer [pyridoxal phosphate (PLP), pyridoxine phosphate (PNP), pyridoxamine phosphate (PMP), pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM)] and 4-pyridoxic acid (4-PA) concentrations and urinary 4-PA levels of 28 white adolescent females, 12-15 years, having radiomonitored plasma PLP concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate status were determined. Mean daily vitamin B-6 and protein intakes of the subjects were 1.48 mg and 78.3 g, respectively. The ranges for plasma B-6 vitamer and 4-PA concentrations for these subjects which had seemingly adequate vitamin B-6 status were as follows: (nmol/L) PLP, 40.9-122.2; PNP, 0-16.1; PMP, 0-8.1; PL, 0- 15.0; PN, 0-21.9; PM, 0-17.8; and 4-PA, 0-55.7. PLP was the predominant plasma B-6 vitamer as well as being the only vitamer found in plasma of all subjects. Urinary 4-PA concentrations of the girls ranged from 0.11-2.50 pmol/mmol creatinine. The B-6 vitamer values of these white adolescent girls should be of use in the establishment of normal ranges for vitamin B-6 status parameters. HPLC methodologies seem to be advantageous for the rapid and accurate assessment of vitamin B-6 status. / Ph. D.

The problem of this study is to determine the vitamin B-6 status of patients who have chronic obstructive pulmonary disease (COPD). Erythrocyte aspartate transaminase assay was the method for measuring vitamin B-6 status. The vitamin B-6 status was examined in thirty subjects (ten COPD subjects and twenty control subjects). An unpaired t-test was used to compare the vitamin B-6 status of the COPD group versus the control group. Four determinants (percentage stimulation, ratio of basal to stimulated activity, basal activity, and stimulated activity) were used to determine vitamin B-6 status in both groups of subjects. Percentage stimulation and ratio of basal to stimulated activity were not significantly different (control group versus COPD group) at the .05 level. However, two of ten COPD subjects had values for percentage stimulation that were two standard deviations above the mean, indicating a poor B-6 status. In contrast, basal activity and stimulated activity of erythrocyte aspartate transaminase were found to be significantly lower at the .05 level in the COPD group than the control group. Therefore, the COPD subjects as a group had some biochemical characteristics of a lower level of vitamin B-6 than the controls.

A acne é uma doença de pele com alta prevalência e seu tratamento é importante para evitar lesões cutâneas permanentes ou o agravamento de transtornos psicológicos provenientes do abalo à autoestima. Dessa forma, o desenvolvimento de formulações dermocosméticas eficazes que possam melhorar as condições desse tipo de pele é de grande valia. Dentre os ativos com potenciais benefícios para o controle de alguns dos principais fatores causadores da acne, estão a vitamina B3, um derivado de vitamina B6 e o PCA zinco. Assim o objetivo deste estudo foi o desenvolvimento de formulações dermocosméticas para a pele acnéica contendo vitamina B3, derivado lipossolúvel de vitamina B6 e PCA zinco bem como a avaliação da estabilidade e eficácia clínica dessas formulações. Para tal, foram desenvolvidas diferentes formulações, as quais, em um primeiro momento, foram avaliadas quanto à estabilidade frente à adição do ingrediente ativo Zinc PCA. Após esta etapa, os demais ingredientes ativos foram adicionados e as formulações foram submetidas a testes preliminares de estabilidade e ao estudo da estabilidade física por determinação do comportamento reológico. A formulação mais estável foi avaliada quanto à compatibilidade cutânea e também em relação à comedogenicidade do veículo. A formulação composta pelos ingredientes ativos foi avaliada, ainda, quanto as suas características sensoriais e eficácia clínica. Os estudos de eficácia foram realizados por meio de métodos objetivos e subjetivos, após seis semanas do uso da formulação. Os métodos objetivos consistiram no uso de metodologias in vivo, não invasivas (métodos biofísicos e de imagem), sendo avaliados parâmetros relacionados à hidratação, função barreira, conteúdo lipídico, pH cutâneo, contagem de porfirinas, de microcomedões e de lesões inflamatórias. Em relação aos métodos subjetivos, foi realizada a percepção da eficácia por meio de um questionário para a comparação da pele antes e após o tratamento. Os resultados mostraram que, de todas as formulações desenvolvidas, apenas uma mostrou-se estável frente aos testes de estabilidade realizados. A formulação (veículo e adicionada de ingredientes ativos) apresentou compatibilidade cutânea considerada como \"muito boa\", de acordo com o teste realizado, e o veículo sem potencial comedogênico. Na avaliação sensorial as frequências obtidas para os parâmetros considerados como ruins foram baixas, indicando que o sensorial da formulação mostrou-se adequado para as finalidades propostas. No estudo de eficácia clínica, a formulação não alterou a hidratação e a função barreira da pele e mostrou-se eficaz na redução da contagem de porfirinas e das lesões inflamatórias (p<0,05). A avaliação clínica por métodos subjetivos mostrou a eficácia da formulação quanto à melhora da acne inflamatória, oleosidade da pele, hidratação e maciez. Por fim, os resultados obtidos mostraram que a formulação desenvolvida é eficaz e compatível com a pele, bem como a importância da pesquisa e desenvolvimento para a obtenção de formulações estáveis, seguras, eficazes e com sensorial adequado. / Acne is a skin disease with high prevalence and its treatment is important to prevent permanent skin lesions or the aggravation of psychological disorders due to self-esteem shaken. This way, the development of effective dermocosmetic formulations, that can improve the conditions of this skin type, is very important. Vitamin B3, a vitamin B6 derivative and zinc PCA are among the active ingredients which present potential benefits in the controlling of some pathogenic factors of acne. Thus, the aim of this research was to develop cosmetic formulations for acneic skin containing vitamin B3, vitamin B6 lipophilic derivative and zinc PCA, as well as the evaluation of stability and clinical efficacy. For this purpose, were developed different formulations which, at first, were evaluated in terms of stability face to zinc PCA addition. After this, the others active ingredients were added and the formulations were submitted to preliminary tests of stability and physical stability studies by rheological behavior determination. The most stable formulation was subject to skin compatibility evaluation and vehicle comedogenicity. The formulation with the active ingredients was also evaluated regarding their sensorial characteristics and clinical efficacy. Efficacy studies were performed by means of objective and subjective methods, after a sixweek- period of use of the formulation. The objective methods consisted in non-invasive in vivo methodologies (biophysical techniques and image analysis) where were evaluated hydration, barrier function, lipid content, skin pH and the counting of porphyrins, microcomedones and inflammatories lesions. In relation to subjective methods, was performed the efficacy perception using a questionnaire in order to compare the skin before and after the treatment. The results showed that among the formulations developed, only one kept stable after the stability tests. The formulations were considered as \"very good\" on skin compatibility test and showed no comedogenic potential. In sensorial evaluation, frequencies obtained for the parameters considered bad were low, which indicate the sensorial of the formulation was adequate for the purposes. In clinical efficacy study, the formulation under study did not alter the parameters related to hydration and skin barrier function and was effective in reducing the counting of porphyrins and inflammatories lesions (p<0,05). Clinical evaluation by subjective methods showed the formulation effectiveness regarding the improvement of inflammatory acne, skin oiliness, hydration and softness. Finally, the results obtained showed the formulation developed is effective and compatible with the skin and, besides the importance of research and development for obtaining stable, safe and effective formulations with suitable sensorial.

O metabolismo de vitamina B9 é um alvo terapêutico conhecido para malária. A vitamina B6 foi validada como essencial para o parasita. Esse trabalho analisa bioquimica- e estruturalmente mutantes da enzima piridoxal quinase, pela combinação de análises in silico associadas a ensaios bioquímicos. A estrutura da PdxK de P. falciparum permitiu a localização do sítio ativo e da interface de dimerização. Logo foram sugeridas mutações que alterassem esses resíduos para investigar sua importância. Dinâmica molecular sugere que a dimerização é responsável pela estabilidade do sítio ativo, algo coerente com a diminuição da atividade enzimática na maioria das mutantes. Filtração em gel aponta um equilíbrio entre a conformação monômero-dímero mostrando que as mutações na interface não estão relacionadas a dimerização, mas envolvidas com a estabilização do folding na região do sítio ativo. A mesma é recoberta por uma tampa que impede a auto-hidrólise do ATP, há também um resíduo serina que estabiliza a conformação do piridoxal durante a catálise, as mutações em ambas as regiões levaram a inativação da enzimática. A hipótese de que a interação da PfPdxK com ligantes de RNA não se mostrou conclusiva. / Vitamin B9 metabolism is a known drug-target for malaria. Vitamin B6 was validated as essential for the parasite. We analysed biochemically and structurally the plasmodial dimeric pyridoxal kinase. PfPdxKs allowed us to determine the localisation of the active site as well the interface between the two monomers and to identify the involved residues. Molecular dynamics shows that the PdxKs dimerization is important for the active sites stability, which was confirmed by the decrease of activity in mutants related to this region. Gel filtration revealed equilibrium of monomer-dimer conformation and therefore the interface mutations decrease in activity might not be directly related to the dimerization processes, but rather to the active site organisation. The active site region shows a serine involved in keeping the pyridoxals conformation during catalyses and the identified lid region covering the ATP binding site is responsible for preventing auto-hydrolysis. Substitution of the respective amino acid to alanine resulted in enzyme inactivation. In silico analysis of the PfPdxK spacer region identified nucleic acid binding sides, however RNA binding experiments failed so far and the possibility of protein-RNA binding remains for elucidation.

Objetivo Examinar a correlação entre fatores dietéticos, obtidos por questionário de freqüência alimentar (QFA) validado, e concentrações séricas ou plasmáticas de homocisteína (hcy), ácido fólico, vitaminas B12 e B6 em mulheres de São Paulo. População e métodos Foram analisados os dados dietéticos de 1.434 mulheres de 21 a 65 anos de um estudo caso-controle sobre consumo alimentar e lesões neoplásicas do colo uterino realizado em três hospitais públicos da cidade de São Paulo, excluindo-se os casos de câncer invasivo. Todas participantes tiveram sua ingestão alimentar usual avaliada por entrevista, usando um QFA, e forneceram amostras sangüíneas em jejum para separação de plasma e soro. Concentrações séricas de ácido fólico e vitamina B12 séricos foram analisadas por técnica de fluoroimunoensaio, enquanto concentrações plasmáticas de hcy e vitamina B6 foram analisadas por Cromatografia Líquida de Alta Performance em fase reversa. Correlações entre ingestão estimada de nutrientes, ajustados pelas calorias totais, e alimentos com as variáveis bioquímicas foram avaliadas em modelos de regressão linear múltiplos, após ajuste para co-variáveis, tais como idade, Índice de Massa Corporal (IMC), estilo de vida (incluindo tabagismo), morbidade ginecológica pregressa ou atual, história obstétrica e uso de anticoncepcional oral. Resultados Embora apenas 6,2% das participantes do estudo tenham apresentado concentrações séricas de ácido fólico abaixo do valor de referência (7 nmol/L), 45,7% e em 97,1% tiveram um consumo estimado de folato inferior a 180 ug/dia e 400 ug/dia, respectivamente. Modelos de regressão múltiplos mostraram correlação positiva entre ácido fólico sérico e ingestão estimada de proteína, ferro, folato, vitaminas B1, B3, B6, A, C e frutas/sucos cítricos e de vegetais verdes, e correlação inversa entre ácido fólico sérico e consumo estimado de gorduras, doces e leite e derivados. Resultados similares foram obtidos após ajuste adicional para fibra da dieta, exceto com consumo de folato e de vegetais verdes, que perderam a significância estatística como preditores independentes das concentrações séricas de ácido fólico. Concentrações séricas de vitamina B12 abaixo do ponto de corte de 148 pmol/L foram observadas em 11,0% da amostra; a maioria delas (70,4%) apresentou ingestão estimada de vitamina B12 igual ou superior à recomendação (2 ug/dia). As concentrações séricas de vitamina B12 foram positivamente correlacionadas com consumo estimado de produtos lácteos e das vitaminas B2 e B12. A ingestão de fibra, vitamina E e leguminosas foi inversamente correlacionada com as concentrações séricas de vitamina B12. Ingestão de vitamina B6 abaixo das recomendações de 1,3 mg/dia (&#8804;50 anos) e 1,5 mg/dia (>50 anos) foi observada em 49% das participantes. Nenhuma correlação foi encontrada entre dados da dieta e concentrações plasmáticas de vitamina B6. As concentrações plasmáticas de hcy foram positivamente correlacionadas com o consumo estimado de carboidratos e doces, e inversamente correlacionadas com o consumo estimado de proteína, colesterol, ferro, zinco de origem animal, vitaminas A, B2, B12 e B6, e pescados. Entretanto, essas correlações perderam a significância após ajuste adicional por proteína da dieta, um dos mais fortes preditores das concentrações plasmáticas de hcy. Conclusão Nutrientes e alimentos selecionados da dieta mostraram-se preditores independentes das concentrações séricas de ácido fólico e de vitamina B12, indicando as principais fontes alimentares desses nutrientes nesta população e em outras similares. A forte correlação negativa entre concentração plasmática de Hcy e proteína da dieta sugere base para o planejamento de futuras intervenções nutricionais. Nenhuma correlação foi observada entre concentração plasmática de vitamina B6 e fatores dietéticos estimados. / Objective To examine whether measurements of dietary intakes, obtained with a validated quantitative food frequency questionnaire (FFQ), correlated with serum or plasma levels of folic acid, vitamins B12 and B6 and homocystein (hcy) measured in low-income women living in São Paulo, Brazil. Population and methods We analyzed dietary data from 1434 women aged 21-65 years enrolled in a case-control study of diet and cervical cancer carried out in three public hospitals of São Paulo. Data for women with invasive cervical cancer were excluded. All participants had their usual dietary intake assessed by interview, using a validated FFQ, and provided a fasting blood sample for serum and plasma separation. Serum concentrations of folic acid and vitamin B12 were measured by fluorimmunoassay, while serum levels of vitamin B6 and plasma levels of hcy were measured by reversed-phase high performance liquid chromatography. Correlations between estimates of food and energy-adjusted nutrient intakes and levels of folic acid, vitamins B12 and B6 and hcy were assessed using multiple linear regression models, adjusted for covariates such as age, body mass index, lifestyle (including smoking), past and current gynecologic morbidity and obstetric history, and use of oral contraceptives. Results Although only 6.2% of the study participants had serum folic acid levels below the reference value of 7 nmol/L, 45.7% and 97.1% had a dietary intake of folic acid estimated to be less than 180 &#61549;g/day and 400 &#61549;g/day, respectively. Multiple linear models showed serum folic acid levels to be positively correlated with the estimated intake of protein, iron, folate, vitamins B1, B3, B6, A and C, citrus fruits and juices and green vegetables, and negatively correlated with the estimated intake of fat, sweets and dairy products. Similar results were obtained after a further adjustment for fiber intake in the model, except for the estimated intake of folic acid and green vegetables, which lost their statistical significance as independent predictors of serum folic acid levels. Serum levels of vitamin B12 below the cut-off point of 148 pmol/L were found in 11.0% of study participants; most of them (70.4%) had their vitamin B12 intake estimated to be equal or greater than the reference value of 2 &#61549;g/day. Serum levels of vitamin B12 were positively correlated with the estimated intake of dairy products and vitamins B2 and B12. The estimated intakes of fiber, vitamin E and beans were negatively correlated with serum levels of vitamin B12. Dietary vitamin B6 was estimated to be below the recommended levels of 1.3 mg/day (age &#61603; 50 years) or 1.5 mg/day (age > 50 years) in 49.0% of study participants. No correlation was found between estimated intakes of foods and nutrients and plasma levels of vitamin B6. Hcy concentrations were positively correlated with the estimated intake of carbohydrates and sweets, and negatively correlated with the estimated intake of protein, cholesterol, iron, zinc of animal origin, vitamins A, B2, B12 and B6 and fishes. However, these correlations were no longer significant after additional adjustment for dietary protein, the strongest predictor of hcy plasma levels. Conclusion The estimated dietary intakes of selected foods and nutrients were shown to be independent predictors of measured serum levels of folic acid and vitamin B12, providing a basis for indentifying the main dietary sources of these nutrients in this and similar populations. The strong negative correlation between plasma levels of hcy and dietary protein provides a basis for future nutritional interventions. No correlation was found between plasma concentrations of vitamin B6 and estimated dietary intakes.