Peptide nucleic acid pre-gel hybridization: An alternative to Southern hybridization

We have found that it is possible to use labeled peptide nucleic acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as an alternative for Southern hybridization. In this technique, the PNA probe is hybridized to a denatured DNA sample at low ionic strength and the mixture is loaded directly on to an electrophoresis system for size separation. Ensuing gel electrophoresis separates the single-stranded DNA fragments by length. The neutral backbone of PNA allows for hybridization at low ionic strength and imparts very low mobility to excess PNA. Detection of the bound PNA is possible by direct fluorescence detection with capillary electrophoresis, or the DNA/PNA hybrids can be blotted onto a membrane and detected with standard chemiluminescent techniques. Efficient single bp discrimination was achieved routinely using both capillary and slab-gel electrophoresis.

Nicotianamine Chelates Both FeIII and FeII. Implications for Metal Transport in Plants1

Nicotianamine (NA) occurs in all plants and chelates metal cations, including FeII, but reportedly not FeIII. However, a comparison of the FeII and ZnII affinity constants of NA and various FeIII-chelating aminocarboxylates suggested that NA should chelate FeIII. High-voltage electrophoresis of the FeNA complex formed in the presence of FeIII showed that the complex had a net charge of 0, consistent with the hexadentate chelation of FeIII. Measurement of the affinity constant for FeIII yielded a value of 1020.6, which is greater than that for the association of NA with FeII (1012.8). However, capillary electrophoresis showed that in the presence of FeII and FeIII, NA preferentially chelates FeII, indicating that the FeIINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H2O2-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2′-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2′-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed.

Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion.

Cholecystokinin (CCK) secretion in rats and humans is inhibited by pancreatic proteases and bile acids in the intestine. It has been hypothesized that the inhibition of CCK release caused by pancreatic proteases is due to proteolytic inactivation of a CCK-releasing peptide present in intestinal secretion. To purify the putative luminal CCK-releasing factor (LCRF), intestinal secretions were collected by perfusing a modified Thiry-Vella fistula of jejunum in conscious rats. From these secretions, the peptide was concentrated by ultrafiltration followed by low-pressure reverse-phase chromatography and purified by reverse-phase high-pressure liquid chromatography. Purity was confirmed by high-performance capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stimulate pancreatic protein secretion when infused into the proximal small intestine of conscious rats. Partially purified fractions strongly stimulated both pancreatic secretion and CCK release while CCK receptor blockade abolished the pancreatic response. Amino acid analysis and mass spectral analysis showed that the purified peptide is composed of 70-75 amino acid residues and has a mass of 8136 Da. Microsequence analysis of LCRF yielded an amino acid sequence for 41 residues as follows: STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV. When infused intraduodenally, the purified peptide stimulated pancreatic protein and fluid secretion in a dose-related manner in conscious rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography using antisera raised to synthetic LCRF-(1-6) abolished the CCK releasing activity of intestinal secretions. These studies demonstrate, to our knowledge, the first chemical characterization of a luminally secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release.

Análise de flavanóides por cromatografia líquida de alta eficiência e eletroforese capilar - otimização de separação e aplicações tecnológicas

No presente trabalho foram estudadas as separações de 18 flavonóides (9 agliconas e 9 glicosídeos) pelas técnicas de Cromatografia Líquida de Alta Eficiência em fase reversa (RP-HPLC) e Cromatografia Micelar Eletrocinética em fluxo reverso (RF-Meck). Em ambas as técnicas foram avaliados solventes puros (metanol, acetonitrila e tetrahidrofurano) e suas misturas como formas de promover a variação de seletividade, através da modificação da fase móvel em HPLC, e da natureza do aditivo orgânico em RF-Meck. Nos estudos efetuados em HPLC utilizando-se gradiente, pode-se comprovar a possibilidade da modelagem do fator de retenção em funçã da proporção de solvente utilizados (MeOH, ACN, THF e suas misturas). Pode-se ainda, com base nos dados de retenção e na análise hierárquica de c1usters, diferenciar quatro diferentes grupos de sistemas cromatográficos com diferentes seletividades para flavonóides agliconas, e outros quatro com diferentes seletividades para glicosídeos. Os sistemas cromatográficos mais ortogonais (cada um pertencente a um grupo de seletividade) foram aplicados na separação de uma planta modelo (Azadirachta indica), de onde pode-se escolher a fase móvel mais seletiva para se otimizar a separação dos flavonóides glicosilados presentes nas folhas desta planta. No método final otimizado pode-se identificar e quantificar cinco dos flavonóides majoritários presentes, sendo três glicosídeos de quercetina (rutina, isoquercitrina e quercitrina) e dois glicosídeos de kaempferol (astragalin e nicotiflorin), em amostras de duas diferentes procedências (Piracicaba-SP e Silvânia-GO). Nos estudos envolvendo a separação dos dezoito flavonóides por RFMEKC pode-se comprovar diferenças significativas de seletividade quando se varia a natureza do solvente orgânico utilizado como aditivo, além de se observar tendências na migração em função das propriedades do solvente adicionado e da estrutura molecular do flavonóide. O solvente de menor eficiência para separação dos flavonóides foi o MeOH. Através da análise dos eletroferogramas obtidos através de um planejamento experimental de misturas, e das trocas de pares críticos observadas nos vários eletrólitos utilizados, obteve-se um método de separação com apenas um par crítico em menos de 12 minutos de corrida. O coeficiente de variação obtido para o fator de retenção foi de 1,5% e para área de 3%, considerando-se cinco injeções. O método desenvolvido foi aplicado com sucesso na identificação dos flavonóides majoritários presentes na planta modelo (Neem), obtendo-se o mesmo resultado do estudo anterior. Como forma de avaliar a concentração de flavonóides totais presentes em espécies vegetais é comum a análise de extratos após hidrólise ácida (conversão de todos glicosídeos em agliconas). Desta forma otimizou-se uma metodologia de separação em RP-HPLC de 8 flavonóides agliconas comumente presentes em alimentos e extratos vegetais de uso cosmético. A otimização foi efetuada mediante um planejamento experimental de misturas, para escolha da fase móvel mais seletiva, e de um planejamento fatorial composto central, para otimização das condições de gradiente. O método obtido foi o mais rápido já visto dentro da literatura consultada. A separação em linha de base foi efetuada em menos de 15 minutos, com coeficientes de variação de área entre 0,1 e 1,8%, coeficiente de correlação de 0,9993 a 0,9994 na faixa de 5 a 100 µg/mL, e limites de quantificação estimados na faixa de 0,1 a 0,21µg/mL. O método desenvolvido foi aplicado na otimização das condições de hidrólise de um extrato de Neem. A otimização foi efetuada através de metodologia de superfície de resposta, levando-se em consideração a concentração de ácido adicionada, o tempo de reação, a temperatura, e a concentração de um antioxidante (ácido ascórbico) adicionado. O resultado da otimização foi uma metodologia de hidrólise com tempo de reação igual a 5 minutos, utilizando-se 1,4 mol/L de HCI, 119°C e 500 µg/mL de ácido ascórbico. Através das metodologias de análise e de hidrólise desenvolvidas pode-se constatar a presença e quantificar no extrato de Neem os flavonóides agliconas quercetina, kaempferol e miricetina. Com o objetivo de se avaliar quais os componentes presentes em extratos vegetais são os responsáveis pelo poder antioxidante atribuído a determinadas plantas, foi montado um sistema de avaliação de poder antioxidante \"on-line\" com reação pós-coluna em HPLC (baseado na literatura) utilizando-se como \"radical livre modelo\" o ABTS. A análise da planta modelo (Neem) neste sistema mostrou que os flavonóides glicosilados identificados nas partes anteriores deste trabalho são os responsáveis pelo poder antioxidante atribuído a esta planta. De posse desta informação, e visando a obtenção de extratos para aplicações cosméticas com poder antioxidante, modelou-se a extração dos flavonóide do Neem em função da composição do solvente extrator (água, etanol , propilenoglicol e suas misturas), de acordo com um planejamento simplex centróide ampliado. Além da previsão da concentração dos princípios ativos pode-se ainda prever outras propriedades dos extratos obtidos, tais como, índice de refração e densidade, muitas vezes constituintes de especificações técnicas de acordo com as aplicações a que se destinam (cremes, xampús, etc).

Avaliação de microtécnicas de extração para análise de lamotrigina em plasma de pacientes epilépticos por eletroforese capilar

A lamotrigina (LTG) é um fármaco pertencente à classe das feniltriazinas utilizado no tratamento de crises epilépticas generalizadas e focais e no tratamento adjunto da epilepsia refratária. Devido à alta variabilidade interindividual, às interações medicamentosas e aos efeitos adversos apresentados durante a administração da LTG, a monitorização terapêutica nos pacientes que fazem uso deste fármaco é necessária para ajuste de dose individual e evitar os efeitos adversos. Assim, o objetivo deste trabalho foi a avaliação de duas técnicas de microextração: a microextração em fase líquida com fibras ocas (HF-LPME) e a microextração líquido-líquido dispersiva (DLLME) para análise da lamotrigina em amostras de plasma de pacientes epilépticos. Primeiramente foram definidas as condições eletroforéticas: foi utilizado um capilar de sílica fundida de 75 ?m de diâmetro interno e 50 cm de comprimento efetivo. O eletrólito de corrida (BGE) foi composto por ácido 2-morfolinoetanosulfônico (MES), na concentração de 130 mmol L-1 e pH 5,0. As análises foram realizadas à temperatura de 20°C e tensão de 15 kV. A amostra foi injetada hidrodinamicamente (0,5 psi por 10 s) e a detecção foi feita em 214 nm. Nestas condições a LTG e o padrão interno (PI), lidocaína, puderam ser analisados em menos de 7 minutos. A HF-LPME foi avaliada no modo de 3 fases, usando 500 ?L de plasma e 3,5 mL de solução fosfato de sódio 50 mmol L-1 pH 9,0 como fase doadora. O solvente utilizado para impregnar a fibra foi o 1-octanol. Como fase aceptora foram utilizados 60 ?L de solução de ácido clorídrico pH 4,0. Para avaliação da DLLME, foi necessária uma etapa de pré-tratamento da amostra (500 ?L de plasma) com 1 mL de acetonitrila. Após isto, 1,3 mL do sobrenadante foram adicionados a 4 mL de solução fosfato de sódio 50 mmol L-1 pH 9,0 e 120 ?L de clorofórmio (solvente extrator) foram injetados nesta amostra aquosa e 165 ?L de fase sedimentada foram recuperados. As características de desempenho analítico para ambos os métodos foram avaliadas, sendo obtida linearidade na faixa de concentração plasmática de 1-20 ?g/mL e limite inferior de quantificação (LIQ) de 1 ?g mL-1. Os ensaios de precisão e exatidão apresentaram valores de acordo com os guias oficiais. Além disso, os métodos foram seletivos, não apresentaram efeito residual e as amostras foram estáveis. Os valores de recuperação foram de 54,3 e 23% para HF-LPME e DLLME, respectivamente. Os métodos validados foram aplicados com sucesso em amostras de plasma de pacientes epilépticos em tratamento com a LTG. Além disso, as duas técnicas foram comparadas e a HF-LPME apresentou vantagens em relação à DLLME, mostrando ser uma técnica promissora para análise de matrizes complexas, com reduzido consumo de solvente orgânico e possibilidade de automação.

Estratégias de microfabricação utilizando toner para produção de dispositivos microfluídicos

Neste trabalho são apresentados processos de microfabricação de estruturas contendo microcanais e sistemas de manipulação hidrodinâmica e eletroosmótica de fluídos. Foram desenvolvidos processos de microfabricação utilizando toner sobre poliéster, toner sobre vidro, toner como resiste, além de métodos alternativos de perfuração de lâminas e selagem de microestruturas em vidro, desenvolvimento de microestruturas para eletroforese capilar e espectrometria de massas com ionização por eletronebulização. A caracterização dos materiais e processos permitiu uma ampla visão das potencialidades e alternativas dos processos de microfabricação, tendo sido demonstrado que os dispositivos produzidos em toner-poliéster são quimicamente resistentes às substâncias tipicamente utilizadas em eletroforese capilar. Neste trabalho, um detector condutométrico sem contato foi implementado em microestruturas de toner-poliéster e a separação eletroforética de alguns metais alcalinos é demonstrada. A microestrutura foi projetada no formato padrão em cruz, tendo o canal de separação 22 mm de comprimento, 12 µm de profundidade e largura típica. A cela condutométrica foi construída sobre o canal de separação utilizando-se fita adesiva de cobre (1 mm de largura) como eletrodos. O sinal aplicado na cela foi de 530 kHz e 10 Vpp . A separação de K+, Na+ e Li+ na concentração de 100 µmol L-1 foi efetuada em torno de 0,8 min, utilizando-se 1 kV como potencial de separação. Foram desenvolvidos microchips para análise por espectrometria de massas com introdução de amostra por eletronebulização, sendo determinado cluster do íon cloreto em concentração de 1 mmol L+. Também solução com 1 mmol/L de glucosamina em água/metanol 1: 1 (v/v), sob corrente de 100 nA gerou sinal estável e livre de descarga corona. Utilizando detecção amperométrica, obteve-se eletroferogramas mostrando a separação de iodeto (10 mmol L-1) e ascorbato (40 mmol L-1) em potencial de separação de 4,0 kV (800 V cm-1 potencial de detecção de 0,9 V (vs. Ag/AgCI), injeção com 1,0 kV/1°s, tampão borato de sódio 10 mmol L+ com CTAH 0,2 mmol L-1, pH 9,2. Obteve-se eficiência de 1,6.104 pratos/m e foi possível obter limites de detecção de 500 nmol L-1 (135 amol) e 1,8 µmol L-1 (486 amol) para iodeto e ascorbato, respectivamente. O processo de fabricação utilizando toner como material estrutural para microchips em vidro foi bem estabelecido, assim como os modos de detecção fotométrico e condutométrico foram demonstrados. Foram obtidos eletroferogramas par detecção condutométrica sem contato de solução 200 µmol L-1 de K+, Na+ e U+, em tampão histidina/ácido lático 30 mmol L-1 9:1 (v/v) água:metanol, injeção eletrocinética de 2,0 kV/5,0 s, potencial de separação de 1 kV, 530 kHz de frequência e tensão de 2,0 Vpp. Também foi implementado um sistema de detecção fotométrico para microchip operando em 660 nm, tendo sido utilizado para a detecção de azul de metileno 1,0 mmol L-1 em tampão de corrida de barato de sódio 20 mmol L-1 (pH 9,2), com o detector posicionado a 40 mm do ponto de injeção e com injeção eletrocinética a 2,0 kV por 12 s com picos bem resolvidos em menos de 1 min.

Biochemical mechanisms involved in pulmonary hypo-alveolarization induced by peroxides contaminating parenteral nutrition in newborn guinea pig

La dysplasie broncho-pulmonaire (DBP), caractérisée par un défaut de l’alvéolarisation, est une complication pathologique associée à un stress oxydant chez le nouveau-né prématuré. La DBP est présente chez près de 50 % des nouveau-nés de moins de 29 semaines de gestation. La nutrition parentérale (NP) que ces nouveau-nés reçoivent pour cause d’immaturité gastro-intestinale est une source importante de stress oxydant. En effet, leur NP est contaminée par des peroxydes, dont l’ascorbylperoxyde qui est une forme peroxydée du déshydroascorbate. La génération des peroxydes est catalysée par la lumière ambiante. La photoprotection de la NP, quoique difficile d’application en clinique, est associée à une diminution de l’incidence de la DBP chez les enfants prématurés. Chez l’animal nouveau-né, la photoprotection de la NP est associée à un meilleur développement alvéolaire. Ainsi, nous émettons l’hypothèse que l’ascorbylperoxide infusé avec la NP cause la perte d’alvéoles suite à une apoptose exagérée induite par l’oxydation du potentiel redox du glutathion. Cette oxydation du potentiel redox serait occasionnée par l’inhibition de la transformation hépatique de la méthionine en cystéine, menant à une diminution de la synthèse de glutathion au foie et dans les tissus tels que les poumons. La confirmation de cette hypothèse suggérera qu’un ajout de glutathion dans la NP permettra une meilleure détoxification de l’ascorbylperoxide par l’action de la glutathion peroxydase, et préviendra l’oxydation du potentiel redox et ainsi, la perte d'alvéoles par apoptose. Objectifs : Le but de mon projet de recherche est de comprendre les mécanismes biochimiques liant la NP et le développement de la DBP chez le nouveau-né prématuré et de proposer une alternative nutritionnelle prévenant le développement de cette complication fréquemment observée dans cette population. Les objectifs spécifiques sont : 1) d’évaluer l’impact, au poumon, de l’infusion de l’ascorbylperoxyde sur l’axe métabolique potentiel redox du glutathion - apoptose - le développement alvéolaire; 2) d’étudier l’impact de l’ascorbylperoxyde et du potentiel redox sur l’activité hépatique de la méthionine adénosyltransférase (MAT), première enzyme de la cascade métabolique transformant la méthionine en cystéine; et 3) de tenter de prévenir l’impact négatif de la NP ou de l’infusion d’ascorbylperoxyde sur le poumon en améliorant le statut en glutathion. Méthodes: Par un cathéter fixé dans la jugulaire, des cochons d’Inde de trois jours de vie (n = 8 par groupe) ont reçu en continu durant 4 jours une NP ou une solution de base (dextrose + NaCl) enrichie des différentes molécules à l’essai. Le premier objectif a été atteint en enrichissant la solution de base en ascorbylperoxyde à 0, 20, 60 et 180 μM. Ces solutions contenaient ou non 350 μM H2O2 pour se rapprocher des conditions cliniques. Le second objectif a été atteint en investiguant les mécanismes d’inhibition de la MAT dans des animaux infusés ou non avec des solutions contenant la solution de base, des peroxydes, du glutathion et la NP (dextrose + acides aminés + multivitamines + lipides). Le troisième objectif a été atteint en ajoutant ou non à une solution d’ascorbylperoxide ou à la NP 10 μM de glutathion (GSSG), afin d’obtenir une concentration plasmatique normale de glutathion. Après 4 jours, les poumons étaient prélevés et traités pour la détermination de GSH et GSSG par électrophorèse capillaire, le potentiel redox était calculé selon l'équation de Nernst et le niveau de caspase-3 actif (marqueur d’apoptose) par Western blot et l’index d’alvéolarisation quantifié par le nombre d’interceptes entre des structures histologiques et une droite calibrée. Les données étaient comparées par ANOVA, les effets étaient considérés comme significatifs si le p était inférieur à 0,05. Résultats: L’infusion de l’ascorbylperoxyde, indépendamment du H2O2, a induit une hypoalvéolarisation, une activation de la caspase-3 et une oxydation du potentiel redox de manière dose-dépendante. Ces effets ont été empêchés par l’ajout de GSSG à la NP ou à la solution d’ascorbylperoxyde (180 M). L’ascorbylperoxyde et le H2O2 ont inhibé l’activité de MAT tandis qu’elle était linéairement modulée par la valeur du potentiel redox hépatique. Conclusion : Nos résultats suggèrent que l’ascorbylperoxyde est l’agent actif de la NP conduisant au développement de la DBP. Ainsi la correction des bas niveaux de glutathion induits par les peroxydes de la NP favorise la détoxification des peroxydes et la correction du potentiel redox pulmonaire ; ce qui a protégé les poumons des effets délétères de la NP en outrepassant l’inhibition de la MAT hépatique. Nos résultats sont d'une grande importance car ils donnent de l'espoir pour une prévention possible de la DBP.

Biochemical mechanisms involved in pulmonary hypo-alveolarization induced by peroxides contaminating parenteral nutrition in newborn guinea pig

La dysplasie broncho-pulmonaire (DBP), caractérisée par un défaut de l’alvéolarisation, est une complication pathologique associée à un stress oxydant chez le nouveau-né prématuré. La DBP est présente chez près de 50 % des nouveau-nés de moins de 29 semaines de gestation. La nutrition parentérale (NP) que ces nouveau-nés reçoivent pour cause d’immaturité gastro-intestinale est une source importante de stress oxydant. En effet, leur NP est contaminée par des peroxydes, dont l’ascorbylperoxyde qui est une forme peroxydée du déshydroascorbate. La génération des peroxydes est catalysée par la lumière ambiante. La photoprotection de la NP, quoique difficile d’application en clinique, est associée à une diminution de l’incidence de la DBP chez les enfants prématurés. Chez l’animal nouveau-né, la photoprotection de la NP est associée à un meilleur développement alvéolaire. Ainsi, nous émettons l’hypothèse que l’ascorbylperoxide infusé avec la NP cause la perte d’alvéoles suite à une apoptose exagérée induite par l’oxydation du potentiel redox du glutathion. Cette oxydation du potentiel redox serait occasionnée par l’inhibition de la transformation hépatique de la méthionine en cystéine, menant à une diminution de la synthèse de glutathion au foie et dans les tissus tels que les poumons. La confirmation de cette hypothèse suggérera qu’un ajout de glutathion dans la NP permettra une meilleure détoxification de l’ascorbylperoxide par l’action de la glutathion peroxydase, et préviendra l’oxydation du potentiel redox et ainsi, la perte d'alvéoles par apoptose. Objectifs : Le but de mon projet de recherche est de comprendre les mécanismes biochimiques liant la NP et le développement de la DBP chez le nouveau-né prématuré et de proposer une alternative nutritionnelle prévenant le développement de cette complication fréquemment observée dans cette population. Les objectifs spécifiques sont : 1) d’évaluer l’impact, au poumon, de l’infusion de l’ascorbylperoxyde sur l’axe métabolique potentiel redox du glutathion - apoptose - le développement alvéolaire; 2) d’étudier l’impact de l’ascorbylperoxyde et du potentiel redox sur l’activité hépatique de la méthionine adénosyltransférase (MAT), première enzyme de la cascade métabolique transformant la méthionine en cystéine; et 3) de tenter de prévenir l’impact négatif de la NP ou de l’infusion d’ascorbylperoxyde sur le poumon en améliorant le statut en glutathion. Méthodes: Par un cathéter fixé dans la jugulaire, des cochons d’Inde de trois jours de vie (n = 8 par groupe) ont reçu en continu durant 4 jours une NP ou une solution de base (dextrose + NaCl) enrichie des différentes molécules à l’essai. Le premier objectif a été atteint en enrichissant la solution de base en ascorbylperoxyde à 0, 20, 60 et 180 μM. Ces solutions contenaient ou non 350 μM H2O2 pour se rapprocher des conditions cliniques. Le second objectif a été atteint en investiguant les mécanismes d’inhibition de la MAT dans des animaux infusés ou non avec des solutions contenant la solution de base, des peroxydes, du glutathion et la NP (dextrose + acides aminés + multivitamines + lipides). Le troisième objectif a été atteint en ajoutant ou non à une solution d’ascorbylperoxide ou à la NP 10 μM de glutathion (GSSG), afin d’obtenir une concentration plasmatique normale de glutathion. Après 4 jours, les poumons étaient prélevés et traités pour la détermination de GSH et GSSG par électrophorèse capillaire, le potentiel redox était calculé selon l'équation de Nernst et le niveau de caspase-3 actif (marqueur d’apoptose) par Western blot et l’index d’alvéolarisation quantifié par le nombre d’interceptes entre des structures histologiques et une droite calibrée. Les données étaient comparées par ANOVA, les effets étaient considérés comme significatifs si le p était inférieur à 0,05. Résultats: L’infusion de l’ascorbylperoxyde, indépendamment du H2O2, a induit une hypoalvéolarisation, une activation de la caspase-3 et une oxydation du potentiel redox de manière dose-dépendante. Ces effets ont été empêchés par l’ajout de GSSG à la NP ou à la solution d’ascorbylperoxyde (180 M). L’ascorbylperoxyde et le H2O2 ont inhibé l’activité de MAT tandis qu’elle était linéairement modulée par la valeur du potentiel redox hépatique. Conclusion : Nos résultats suggèrent que l’ascorbylperoxyde est l’agent actif de la NP conduisant au développement de la DBP. Ainsi la correction des bas niveaux de glutathion induits par les peroxydes de la NP favorise la détoxification des peroxydes et la correction du potentiel redox pulmonaire ; ce qui a protégé les poumons des effets délétères de la NP en outrepassant l’inhibition de la MAT hépatique. Nos résultats sont d'une grande importance car ils donnent de l'espoir pour une prévention possible de la DBP.

Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis

Aims: Isolation, identification and characterization of a highly efficient isomaltulose producer. Methods and Results: After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis. An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity. Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90-100 U mg(-1) DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41-66 U mg(-1) DW) or a reference strain of Erwinia rhapontici (0.3-0.9 U mg(-1) DW). Maximum yield of isomaltulose at 78-80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe. UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose. Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype. Significance and Impact of Study: This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose.

Urinary proteomic biomarkers in coronary artery disease

Urinary proteomics is emerging as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases. We tested whether signatures of urinary polypeptides can contribute to the existing biomarkers for coronary artery disease (CAD). We examined a total of 359 urine samples from 88 patients with severe CAD and 282 controls. Spot urine was analyzed using capillary electrophoresis on-line coupled to ESI-TOF-MS enabling characterization of more than 1000 polypeptides per sample. In a first step a "training set" for biomarker definition was created. Multiple biomarker patterns clearly distinguished healthy controls from CAD patients, and we extracted 15 peptides that define a characteristic CAD signature panel. In a second step, the ability of the CAD-specific panel to predict the presence of CAD was evaluated in a blinded study using a "test set." The signature panel showed sensitivity of 98% (95% confidence interval, 88.7-99.6) and 83% specificity (95% confidence interval, 51.6-97.4). Furthermore the peptide pattern significantly changed toward the healthy signature correlating with the level of physical activity after therapeutic intervention. Our results show that urinary proteomics can identify CAD patients with high confidence and might also play a role in monitoring the effects of therapeutic interventions. The workflow is amenable to clinical routine testing suggesting that non-invasive proteomics analysis can become a valuable addition to other biomarkers used in cardiovascular risk assessment.

Comparative performance of analytical techniques for polyhexamethylene biguanide (PHMB) in ophthalmic solutions

Purpose: The use of PHMB as a disinfectant in contact lens multipurpose solutions has been at the centre of much debate in recent times, particularly in relation to the issue of solution induced corneal staining. Clinical studies have been carried out which suggest different effects with individual contact lens materials used in combination with specific PHMB containing care regimes. There does not appear to be, however, a reliable analytical technique that would detect and quantify with any degree of accuracy the specific levels of PHMB that are taken up and released from individual solutions by the various contact lens materials. Methods: PHMB is a mixture of positively charged polymer units of varying molecular weight that has maximum absorbance wavelength of 236 nm. On the basis of these properties a range of assays including capillary electrophoresis, HPLC, a nickelnioxime colorimetric technique, mass spectrophotometry, UV spectroscopy and ion chromatography were assessed paying particular attention to each of their constraints and detection levels. Particular interest was focused on the relative advantage of contactless conductivity compared to UV and mass spectrometry detection in capillary electrophoresis (CE). This study provides an overview of the comparative performance of these techniques. Results: The UV absorbance of PHMB solutions, ranging from 0.0625 to 50 ppm was measured at 236 nm. Within this range the calibration curve appears to be linear however, absorption values below 1 ppm (0.0001%) were extremely difficult to reproduce. The concentration of PHMB in solutions is in the range of 0.0002–0.00005% and our investigations suggest that levels of PHMB below 0.0001% (levels encountered in uptake and release studies) can not be accurately estimated, in particular when analysing complex lens care solutions which can contain competitively absorbing, and thus interfering, species in the solution. The use of separative methodologies, such as CE using UV detection alone is similarly limited. Alternative techniques including contactless conductivity detection offer greater discrimination in complex solutions together with the opportunity for dual channel detection. Preliminary results achieved by TraceDec1 contactless conductivity detection, (Gain 150%, Offset 150) in conjunction with the Agilent capillary electrophoresis system using a bare fused silica capillary (extended light path, 50 mid, total length 64.5 cm, effective length 56 cm) and a cationic buffer at pH 3.2, exhibit great potential with reproducible PHMB split peaks. Conclusions: PHMB-based solutions are commonly associated with the potential to invoke corneal staining in combination with certain contact lens materials. However this terminology ‘PHMBbased solution’ is used primarily because PHMB itself has yet to be adequately implicated as the causative agent of the staining and compromised corneal cell integrity. The lack of well characterised adequately sensitive assays, coupled with the range of additional components that characterise individual care solutions pose a major barrier to the investigation of PHMB interactions in the lenswearing eye.

Design, synthesis and biological evaluation of potential antibacterial oligonucleotides

Purine and pyrimidine triplex-forming oligonucleotides (TFOs), as potential antibacterial agents, were designed to bind by Hoogsteen and reverse Hoogsteen hydrogen bonds in a sequence specific manner in the major groove of genomic DNA at specific polypurine sites within the gyrA gene of E. coli and S. pneumoniae. Sequences were prepared by automated synthesis, with purification and characterisation determined by high performance liquid chromatograpy, capillary electrophoresis and mass spectrometry. Triplex stability was assessed using melting curves where the binding of the third strand to the duplex target, was assessed over a temperature range of 0-80°C, and at pH 6.4 and 7.2. The most successful of the unmodified TFOs (6) showed a Tm value of 26 °C at both pH values with binding via reverse Hoogsteen bonds. Binding to genomic DNA was also demonstrated by spectrofluorimetry, using fluorescein-labelled TFOs, from which dissociation constants were determined. Modifications in the form of 5mC, 5' acridine attachment, phosphorothioation, 2'-0-methylation and phosphoramidation, were made in order to. increase Tm values. Phosphoramidate modification was the most with increased Tm values of 42°C. However, the final purity of these sequences was poor due to their difficult syntheses. FACS (fluorescent activated cell sorting) analysis was used to determine the potential uptake of a fluorescently labelled analogue of 6 via passive, coJd shock mediated, and anionic liposome aided, uptake. This was established at 20°C and 37°C. At both temperatures anionic lipid-mediated uptake produced unrivalled fluorescence, equivalent to 20 and 43% at 20 and 37°C respectively. Antibacterial activity of each oligonucleotide was assessed by viable count anaJysis relying on passive uptake, cold shocking techniques, chlorpromazine-mediated uptake, and, cationic and anionic lipid-aided uptake. All oligonucleotides were assessed for their ability to enhance uptake, which is a major barrier to the effectiveness of these agents. Compound 6 under cold shocking conditions produced the greatest consistent decline in colony forming units per ml. Results for this compound were sometimes variable indicating inconsistent uptake by this particular assay method.

Improved estimation of postmortem interval with Cardiac Troponin T and improved analytical methods

Accurate knowledge of the time since death, or postmortem interval (PMI), has enormous legal, criminological, and psychological impact. In this study, an investigation was made to determine whether the relationship between the degradation of the human cardiac structure protein Cardiac Troponin T and PMI could be used as an indicator of time since death, thus providing a rapid, high resolution, sensitive, and automated methodology for the determination of PMI. ^ The use of Cardiac Troponin T (cTnT), a protein found in heart tissue, as a selective marker for cardiac muscle damage has shown great promise in the determination of PMI. An optimized conventional immunoassay method was developed to quantify intact and fragmented cTnT. A small sample of cardiac tissue, which is less affected than other tissues by external factors, was taken, homogenized, extracted with magnetic microparticles, separated by SDS-PAGE, and visualized with Western blot by probing with monoclonal antibody against cTnT. This step was followed by labeling and available scanners. This conventional immunoassay provides a proper detection and quantitation of cTnT protein in cardiac tissue as a complex matrix; however, this method does not provide the analyst with immediate results. Therefore, a competitive separation method using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was developed to study the interaction between human cTnT protein and monoclonal anti-TroponinT antibody. ^ Analysis of the results revealed a linear relationship between the percent of degraded cTnT and the log of the PMI, indicating that intact cTnT could be detected in human heart tissue up to 10 days postmortem at room temperature and beyond two weeks at 4C. The data presented demonstrates that this technique can provide an extended time range during which PMI can be more accurately estimated as compared to currently used methods. The data demonstrates that this technique represents a major advance in time of death determination through a fast and reliable, semi-quantitative measurement of a biochemical marker from an organ protected from outside factors. ^

Synthesis & retention mechanisms of novel mixed mode stationary phase for detection of specific polar low molecular weight biological samples

The thesis primarily reports the synthesis, characterization and application of novel mixed mode stationary phases for Hydrophilic Interaction Liquid Chromatography (HILIC). HILIC is a rapidly emerging chromatographic mode that is finding great applicability in the analysis of polar organic molecules. In addition, there is a chapter on the analysis of Bisphenol A and related species using capillary electrophoresis (CE) coupled with boron-doped diamond electrodes for electrochemical detection. The synthesis and characterization of the novel mixed mode stationary phases prepared in this work is an important contribution to the field as the materials prepared exhibited better performance than similar materials obtained commercially. In addition a more thorough characterization of the materials (e.g.,thermogravimetric analysis, various NMR modes, elemental analysis, etc.) and resulting columns (e.g., H) than is typically encountered. The application of these new materials to the analysis of sugars using evaporative light scattering is also novel. In CE studies, electrochemical detection is sufficiently rare that the work is also novel.