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 (≤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 g/day and 400 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 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  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.

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.

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.

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.

Vitamin B-6 status was examined in a group of 46 elderly subjects who were selected from nursing home residents, hospital patients, and free living individuals in Denton County. Subjects were limited to men and women over 60 years of age. Erythrocyte aspartate aminotransferase stimulation with pyridoxal phosphate (in-vitro) was studied as the biochemical criterion of vitamin B-6 status. The pyridoxine status of these 46 subjects (the reference group) was measured in order to be able to identify people with a relatively poor B-6 status. A sub-group of the reference group was composed of 4 subjects who took B-6 supplements (supplemented group). There was no significant difference (0.05 level) in the basal activity, stimulated activity, percentage stimulation, or body weight, after treatment with 10 mg pyridoxine hydrochloride for 4 weeks, even though all 4 subjects had an improved B-6 status (based on percentage stimulation) after taking the supplement. The data indicated that of the 4 subjects tested, 2 showed a large change in the basal activity, stimulated activity, and percentage stimulation. The lack of significant difference (0.05 level) was probably due to a small sample size. One subject reported an increased appetite and body weight after treatment with pyridoxine.

Pyridoxal 5'-phosphate (PLP) is the cofactor for over 140 vitamin B6 (PLP)-dependent enzymes that are involved in various metabolic and biosynthetic pathways. Pyridoxal kinase (PL kinase) and pyridoxine 5’-phosphate oxidase (PNP oxidase) are the two key enzymes that metabolize nutritional forms of vitamin B6, including pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM) to the active cofactor form, PLP. Disruption of the PLP metabolic pathway due to mutations in PNP oxidase or PL kinase result in PLP deficiency, which is implicated in several neurological pathologies. Several ingested compounds are also known to result in PLP deficiency with concomitant neurotoxic effects. How these mutations and compounds affect B6 metabolism is not clearly understood. On the other hand, an emerging health problem is the intake of too much vitamin B6 as high doses of the reactive PLP in the cell exhibits toxic effects, including sensory and motor neuropathies. The overall aim of this research is to understand the catalytic function of PL kinase and the regulatory pathway of PLP metabolism. Using site-directed mutagenesis (Asp235Asn, Asp235Ala), kinetic and structural studies, we have shown that Asp235 may play a catalytic role in PL kinase phosphorylation activity. We also show that human PL kinase binds its substrates, PL and MgATP synergistically, and that the enzyme requires Na+ (or K+) and Mg2+ for its activity. Using kinetic study, we show severe induced MgATP substrate inhibition of PL kinase in the presence of its product, PLP, and we postulate this to be due to the formation of a non-productive ternary complex (Enzyme•PLP•MgATP). Consistently, our crystal structure of human PL kinase (2.1 Å) co-crystallized with MgATP and PLP showed both MgATP and PLP trapped at the active site. Our hypothesis is that this abortive ternary complex might be a physiological process, and that PL kinase uses this mechanism to self-regulate its activity. Our inhibition studies show theophylline, a bronchodilator as a mixed competitive inhibitor of human PL kinase with Ki of 71 μM. Our structural study (2.1 Å) shows theophylline bound at the substrate, PL binding site of human PL kinase. We also identified several potential PL kinase inhibitors from the DrugBank Chemical Compound database. Some of these compounds, including enprofylline, theobromine, caffeine, and lamotrigine, which incidentally exhibit similar neurotoxic effects as theophylline, show significant inhibitory effect on human PL kinase. Further studies are also planned to investigate the effect of these drugs on vitamin B6 metabolism in vivo.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

The study begins with an account of Blake, as voiced by Allen Ginsberg, taking part in a key Sixties anti-war protest, and goes on to examine some theoretical aspects of Blake's relationship with the Sixties. In Chapter One, I explore the relationship between ‘popular Blake', ‘academic Blake', and ‘countercultural Blake'. The chapter seeks to provide a revisionist account of the relationship between Blake's Sixties popularity and his earlier reception, suggesting that all three elements of Blake's Sixties reception – popular, academic and countercultural – have long been intertwined, and continue to interact in the Sixties themselves. In Chapters Two and Three, I focus in detail on Allen Ginsberg as a central figure not only in Blake's countercultural popularization, but also in the creation of Sixties counterculture itself. The first of these chapters, ‘Visionary Blake, Physical Blake, Psychedelic Blake', looks in detail at Ginsberg's 1948 ‘Blake vision' and the way Ginsberg later uses it to construct a Blakean narrative for the Sixties. I examine the significant differences between the versions of this event presented in Ginsberg's early poems and in his later prose and interview accounts, and Ginsberg's consequent attempts to develop a general theory of poetry in which the specific effects of Blake's poetry on the consciousness are compared to those of psychedelic drugs. Finally, I suggest that there are analogies between this ‘psychedelic' approach to Blake and the interest that Aldous Huxley had in using psychedelics to access Blake's own visionary state of consciousness. Chapter Three, ‘Ginsberg's Blakean Albion', analyses a selection of Ginsberg's poems, all linked to Blake's myth of Albion. I use these poems to examine the tensions present within the three-way relationship between Blake, Ginsberg and British counterculture. Particular attention is given to Ginsberg's poem ‘Wales Visitation' (1967), a work which I suggest is founded on the joint Romantic inheritance of Blake and Wordsworth, and which demonstrates the ways in which various strands of British Romanticism interact both within Ginsberg's poetry and within the broader Sixties counterculture. The final chapter of the study examines various aspects of the relationship between Blake and Bob Dylan, demonstrating the extent of Blake's influence on Dylan, but also tackling the surprisingly complicated and problematic question of the route(s) by which Blake arrives in Dylan's work.

Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.