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Studium interakce vybraných chemopreventivních sloučenin a potravních karcinogenů s cytochromy P450 / Study on the interaction of chemopreventive compounds and food born carcinogens with cytochrome P450 enzymesBrabencová, Eliška January 2013 (has links)
The use of food supplements containing natural chemopreventive compounds increased in recent years. Some of the most popular chemopreventive compounds are flavonoids. Due to their natural origin, flavonoids are generally accepted as safe compounds. They exert antioxidant, anti-cancer and anti-inflammatory properties. However, flavonoids should be considered as foreign compounds (xenobiotics). Flavonoids interact with many enzymes, among the most important belong cytochromes P450 (CYPs), key enzymes of the first phase of biotransformation of xenobiotics (e.g. drugs, carcinogens). CYPs catalyze reactions leading mainly to detoxification of xenobiotics. However, some CYPs are involved in the activation of carcinogens, particularly CYP1A1 and CYP1A2 activate e.g. heterocyclic amines. Flavonoids might enhance the activation of carcinogens via induction of these CYPs or stimulation of their activities and hence, increase the risk of a cancer development. The thesis is focused on the influence of flavonoids and food carcinogens on the induction and activity of CYP1A1 and CYP1A2 in liver and small intestine of rats. For the purpose of this study, the small intestine was dissected into three parts: proximal (nearest to stomach), middle and distal. Western blotting was used for the evaluation of CYP...
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Studium účinku protinádorových léčiv inhibitorů tyrosinkinas ve formě nanotransportérů / Study of action of anticancer drugs tyrosine kinase inhibitors in a form of nanotransportersTakácsová, Paulína January 2019 (has links)
Tyrosine kinase inhibitors (TKI) are small organic molecules designed for the targeted cancer therapy. They perform the inhibition of activated receptor tyrosine kinases in tumor cells, that defeats tumor growth, proliferation, metastasis and angiogenesis in tumor tissue. Two TKI, lenvatinib and vandetanib, are used in thyroid cancer treatment. This thesis investigates the ways leading to enhancement of efficiency of these anticancer drugs for therapy. One of the studied anticancer drug - lenvatinib - was investigated to be prepared in a nanoform. Nanoparticles were based on protein apoferritin as well as on lipids. Theoretical model of lenvatinib interaction with an apoferritin cavity, as well as the model of its encapsulation obtained by computer modeling indicated that lenvatinib seems not to be suitable for preparation of apoferritin nanoparticles. Since lenvatinib occurs in its neutral form during preparation of nanoparticles, it does not interact with nanoparticle. The unsuccessful experimental preparation of lenvatinib-loaded apoferritin nanoparticles confirmed that lenvatinib is not suitable for its preparation. However, the theoretical model can serve for screening of other potentially suitable drugs before the experimental nanoparticle preparation. Since the experimental preparation of...
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Vliv chemopreventivních látek na cytochromy P450 / Effects of chemopreventive compounds on cytochrome P450sKřížková, Jitka January 2010 (has links)
CHARLES UNIVERSITY IN PRAGUE Faculty of Science Department of Biochemistry Effects of chemopreventive compounds on cytochrome P450s Summary of Ph.D. Thesis RNDr. Jitka Křížková Supervisor: prof. RNDr. Petr Hodek, CSc. Prague 2010 Introduction 1 Introduction According to the World Health Organization statistics, cancer is one of the leading causes of death in the human population worldwide for more than 50 years. Moreover, colorectal and gastrointestinal tract cancers are one of the main types of cancer leading to overall cancer mortality. Prevention consisting in a healthy lifestyle and a natural diet is suggested to be one of the main approaches to reduce cancer risk. In recent years, the consumption and use of dietary supplements containing concentrated chemopreventive phytochemicals increased dramatically. Flavonoids, as the most popular representatives of chemopreventive compounds, present in foods (fruits, vegetables, herbs, beverages) and dietary supplements have the potential to modulate the activity of xenobiotic-metabolizing enzymes [Hodek et al., 2002]. Among proteins interacting with flavonoids, cytochrome P450s (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens), play the most prominent role. The two members of CYP1A subfamily, CYP1A1 and CYP1A2, are involved in the...
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Vliv vandetanibu, lenvatinibu a ellipticinu na expresi potkaních cytochromů P450 1A a 3A / The effect of vandetanib, lenvatinib and ellipticine on the expression of rat cytochromes P450 1A and 3AJelínková, Sandra January 2018 (has links)
In recent years, the inhibiition of tyrosine kinases,which may incorrectly regulate some singaling pathway has been used to treat cancer as so-called biological therapy. An example of such inhibitors are vandetanib and lenvatinib. These two substances are used to treat thyroid gland tumors because they affect vascular growth factor receptor or endothelial growth factor receptor that can regulate tumor growth and metastasis. Ellipticine, which has anti-tumor effects on lots of tumor disease, has been investigated in this study together with vandetanib and lenvatinib. In this diploma thesis, the effect of mentioned tyrosine kinase inhibitors, ellipticine and their combinations on gene and protein expression of CYP1A1, 1A2, 3A1 and 3A2 in rat liver in vivo was determined. Protein expression was studied using Western blot method with imunodetection. Gene expression was assessed by quantitative PCR. Moreover, the effect of tested substances and their combinations on CYP1A activity (measured as 7-ethoxyresorufin O-deethylation), CYP1A2 activity (measured as 7-methoxyresorufin O-demethylation), CYP1A1 activity (measured as Sudan I oxidation), CYP3A specific activity (measured as testosteron 6β-hydroxylation) and ellipticine, vandetanib, lenvatinib metabolism was determined. It has been confirmed that...
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Avaliação da capacidade esteroidogênica dos embriões de preá (Galea spixii): imunomarcação e expressão gênica das enzimas do complexo citocromo P450 / Steroidogenic capacity evaluation of spix yellow-toothed cavy embryos (Galea spixii): immunostaining and gene expression of cytochrome P450 complex enzymesOliveira, Franceliusa Delys de 04 November 2016 (has links)
A síntese dos hormônios esteroides é feita através da ação de um complexo enzimático chamado citocromo P450. As enzimas 3β-HSD (3β-hidroxiesteroide desidrogenase), P450c17 (citocromo P450 17α-hidroxilase/17,20-liase) e P450arom (citocromo P450 aromatase), fazem parte desse complexo e são responsáveis pela síntese dos hormônios progestágenos, andrógenos e estrógenos, espectivamente. Essas enzimas já foram identificadas em inúmeros tecidos, inclusive em embriões em período pré-implantação. Por isso, acredita-se que o blastocisto seja uma fonte de produção de progesterona e estrógeno, hormônios essenciais para implantação e manutenção da gestação. Em roedores, as informações sobre a capacidade esteroidogênica dos embriões são controversas e também restritas apenas a estudos com espécies laboratoriais. Para trazer mais informações sobre a capacidade esteroidogênica em blastocistos de roedores utilizamos o preá (Galea spixii), uma espécie de roedor silvestre encontrada nas regiões de Caatinga e Semiárido do Nordeste brasileiro e muito utilizado na alimentação de famílias de baixa renda como fonte proteica. Diante o exposto, o objetivo deste trabalho é fazer um apanhado histórico sobre os dados publicados a cerca da capacidade dos embriões de diversas espécies de produzir os hormônios esteroides além de identificar e quantificar a expressão das enzimas esteroidogênicas em embriões de preá através das técnicas de imunohistoquímica e PCR em tempo real. As análises morfológicas indicam que o desenvolvimento inicial do preá se assemelha com os demais roedores, porém o tempo de desenvolvimento difere das espécies usadas em laboratório. As marcações da imunohistoquímica foram positivas nos tecidos maternos, mas os embriões não tiveram marcação para nenhuma das três enzimas do estudo. No entanto, os dados obtidos pelo PCR indicam que os genes CYP11, CYP17 e CYP19 foram expressos nos embriões e, portanto, o concepto de G. spixii tem capacidade de produzir hormônios esteroides, assim como visto em várias outras espécies que já foram estudadas / The synthesis of steroid hormones is made through the action of an enzyme complex called cytochrome P450. 3β-HSD (3β-hydroxysteroid dehydrogenase), P450c17 (cytochrome P450 17 α-hydroxylase/17,20-lyase) and P450arom (cytochrome P450 aromatase), are present in this complex and are are responsible for the synthesis of progestins, androgens and estrogens hormones, respectively. These enzymes have been identified in numerous tissues, including embryos in pre-implantation period. Therefore, it is believed that the blastocyst is a source of production of progesterone and estrogen, hormones essential for the implementation and maintenance of pregnancy. In rodents, the information about the steroidogenic capacity of embryos are controversial and confined to studies with laboratory species. To get more information about the steroidogenic capacity in blastocysts of rodents we used the spix yellow-toothed cavy (Galea spixii), a species of wild rodent found in the northeastern of Brazil, and used in the feeding of low-income families as a source of protein. On the above, the aim of this work is to make a historic about the data published about the ability of embryos of various species to produce steroid hormones and identify and quantify the expression of steroidogenics enzymes in embryos of spix yellow-toothed cavy by immunohistochemical and real-time PCR techniques. The morphological analyses indicate that the G. spixii early development resembles other rodents, but development time differs from the species used in laboratory. The immunohistochemical stainning was positive in maternal tissues, but the embryos did not have stainnig for any of the three enzymes of the study. However, the data obtained by PCR indicate that CYP11, CYP17 and CYP19 genes were expressed in embryos and, therefore, the concept of G. spixii has capacity to produce steroid hormones, as seen in several other species that have been studied
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Mecanismos moleculares envolvidos na resposta anti-tumoral e resistência a atra / Molecular mechanisms envolved in the anti-tumoral response and resistance to atRAHasegawa, Ana Paula Guedes 30 September 2005 (has links)
Os gliomas representam o tipo de tumor primário mais comum do sistema nervoso central. Apesar dos avanços nas técnicas cirúrgicas e de radioterapia e nos protocolos de quimioterapia, não há tratamento eficiente disponível. O ácido retinóico na forma all-trans (atRA) é um agente anti-proliferativo utilizado para o tratamento de alguns tipos de lesões pré-malígnas e tumores, como a leucemia promielocítica. Entretanto, sua utilização no tratamento clínico de tumores sólidos, incluindo os gliomas, é limitada, devido ao rápido desenvolvimento de resistência aos efeitos anti-tumorais do atRA. Para endereçar este problema, foi proposto: a) clonar e analisar a função de cyp26b1 de rato, uma enzima da família dos citocromos P450 envolvida na metabolização de ácido retinóico, previamente descrito em nosso laboratório como potencialmente envolvido na resposta de células de glioma ao tratamento com atRA; b) gerar e caracterizar uma variante da linhagem celular C6 de glioma de rato resistente aos efeitos anti proliferativos de atRA. O cDNA de cyp26b1 de rato, até então desconhecido, foi amplificado, clonado e seqüenciado com sucesso. A seqüência protéica correspondente é conservada e agrupa-se no dendrograma com outras proteínas Cyp26B1, mesmo de organismos distantes como zebrafish, em detrimento de outras proteínas parálogas da família Cyp26. Cyp26b1 é fortemente induzida por atRA em células de glioma de rato e humano, de forma dose-dependente. Embora seja expresso em amostras de cérebros humanos normais e de glioma, não foram encontradas diferenças significativas entre os diferentes graus de matlignidade tumoral, ou em comparação com cérebro normal. A super-expressão de cyp26b1 de rato através de transfecção estável de células de glioma de rato e humano, bem como de células P19 de teratocarcinoma de camundongo, não alterou significativamente as características de crescimento celular, tanto em substrato sólido quanto em substrato semi-sólido. O tratamento prolongado de células C6 de glioma de rato com atRA levou à obtenção de uma população policlonal resistente aos efeitos anti-proliferativos de atRA, a partir da qual uma linhagem celular clonal resistente foi isolada com sucesso. Esta variante clonal, denominada linhagem celular C6R, apresenta inibição de crescimento por atRA diminuída, quando comparada com a linhagem celular C6 parental e com a variante ST1. Além disso, esta variante também mostrou uma tendência, embora não significativa estatisticamente, de gerar tumores maiores quando injetados no subcutâneo de camundongos do tipo nude. A expressão de genes previamente descritos como induzidos pelo tratamento com atRA em células ST1 é fortemente inibida na linhagem celular C6R. Além disso, menor expressão de RARβ, RARγ e CRABP-1 foi também observada em células C6R, enquanto que expressão muito maior de RXRγ e CRABP-2 foi encontrada nas células resistentes a atRA, em comparação com as células C6 parentais. Como conclusões gerais deste trabalho, foi proposto que cyp26b1 parece estar envolvido no metabolismo e detoxificação de atRA e parece não ser efetor da inibição de crescimento induzida por atRA, nem estar relacionado com a resistência de células de glioma ao tratamento com atRA. Por outro lado, o isolamento e caracterização de uma linhagem celular resistente ao tratamento com atRA sugere que a resistência está relacionada à expressão diferencial de receptores de retinóides e de proteínas de ligação ao ácido retinóico. / Gliomas are among the most common primary tumors of the central nervous system. Despite the advances in surgical and radiotherapy procedures and chemotherapy protocols, efficient treatment is still not available. Retinoic acid is a anti-proliferative agent used for treatment of a number of pre-malignant lesions and tumors, as promyelocytic leukemia. However, its clinical use in treatment of solid tumors, including gliomas, is impaired by the rapid development of resistance to the anti-tumoral effects of atRA. In order to address this problem, we proposed: a) to clone and analyze the role of rat cyp26b1, a member of cytochrome P450 superfamily envolved in the metabolization of retinoic acid, which has previously been described by our group as being potentially involved in the response of glioma cells to retinoic acid treatment; b) to generate and characterize a variant of rat C6 glioma cell line resistant to anti proliferative effects of atRA. The previously unknown cDNA of rat cyp26b1 was successfully amplified, cloned and sequenced. The protein is conserved and clustered in dendrograms with other cyp26b1 proteins, even from distant organisms as zebrafish, in detriment of other cyp26 paralogs. Cyp26b1 is strongly induced by atRA in rat and human glioma cells, in a dose-dependent fashion. Although expressed in human normal brains and glioma samples, no significant differences were found among different tumor grades nor comparing to normal brain. Stable-transfection and overexpression of rat cyp26b1 in rat and human glioma cell lines, as well as P19 mouse teratocarcinoma cell line, did not significantly modified cell growth properties, either on solid or semi-solid substrates. Prolonged treatment of C6 rat glioma cell line with atRA leads to isolation of an atRA-resistant polyclonal cell population, from which a resistant clonal cell line was successfully isolated. This clonal variant, named C6R cell line, displayed diminished growth inhibition by atRA compared to the parental C6 cell line and the variant ST1 cell line. In addition, this variant also showed a trend, although not quite statistically significant, to generate larger tumors when xenografted s.c. into nude mice. Expression of genes previously described as being induced by atRA-treatment in ST1 cells is strongly impaired in the C6R resistant cell line. In addition, lower expression of RARβ, RARγ and CRABP-1 was also observed in C6R cells, whereas a much higher expression of RXRγ and CRABP-2 was found in the resistant cells when compared to the parental C6 cells. As general conclusions of this work, we found that cyp26b1 is more likely to be involved in primary atRA metabolism and detoxification and does not seem to be an effector of atRA-induced cell growth arrest nor to be related to the resistance of glioma cells to atRA treatment. On the other hand, isolation and characterization of an atRA-resistant cell line suggests that atRA resistance is more likely to be due to differential expression of retinoid receptors and retinoic acid binding proteins.
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Cytochrome P450 et inflammation : approche pharmacogénomique et aspects moleculaires des effets anti-inflammatoires des thiénopyridines / Cytochrome P450 and inflammation : pharmacogenomic approach and molecular aspects of anti-inflammatory effects of theinopyridinesShahabi, Payman 12 September 2013 (has links)
Cette thèse est dédiée à l'approche pharmacogénétique des effets anti-inflammatoires de la thérapie par les thiénopyridines. Prenant en compte que les plaquettes activées jouent un rôle central dans les états inflammatoires et que des polymorphismes du cytochrome P450 (CYP) 2C19 ont été montré responsable de différences inter individuelle dans la réponse de l'effet antiplaquettaire de thiénopyridines, nous avons émis l'hypothèse que CYP2C19 *2 ou *17 sont également associés à la variabilité interindividuelle du potentiel antiinflammatoire des thiénopyridines. Les marqueurs d'inflammation utilisés pour suivre l'effet des thiénopyridines sont : la CRP, l'haptoglobines et l'orosomucoïde. Nous avons démontré que pour interpréter les valeurs de l'haptoglobine il était nécessaire de tenir compte du statut génétique et obtenir des valeurs de référence stratifiés. D'abord dans une population saine, nous n'avons pas trouvé d'association entre les valeurs de base des marqueurs inflammatoires et les polymorphismes fréquents de CYP époxygenases. Dans une population après intervention coronarienne percutanée qui était composée de 1128 sujets traités par clopidogrel ou prasugrel, le niveau de CRP observé a montré une interaction significative entre le tabac et le polymorphisme de CYP 2C19 ; cet effet est indépendant du niveau d'agrégation plaquettaire. Dans une 3ème population, sur plus de 1000 sujets hospitalisés à Coimbra, nous avons identifié une interaction entre le clopidogrel CYP2C19 et les médicaments bloqueurs des canaux calciques. En résumé, tous ces résultats obtenus sur plusieurs populations laissent envisager que les marqueurs d'inflammation pourraient être un moyen intéressant de suivi des patients lors de la thérapeutique par les thiénopyridines / The main part of the thesis is devoted to pharmacogenetic approach to the anti-inflammatory effects of thienopyridine therapy. Taking into the account that activated platelets play a central role in the inflammatory responses and that CYP2C19 gain- and loss-of-function polymorphisms (*2 and *17) are sources of inter-individual difference in response to the anti-platelet effects of thienopyridines, we hypothesized that *2 and/or *17 alleles are also associated with inter-individual variability in the potential inflammation-reducing effects of thienopyridines. The following markers were used to test the hypothesis: CRP, haptoglobin and orosomucoid acid. To be reliably interpretable in daily medical practice, genetic status should be considered for partitioning the reference values of haptoglobin. In a small healthy population, no significant association was observed between *2 allele and changes in levels of inflammatory markers from baseline to 7 days after administration of clopidogrel and our findings did not support the notion that the genetic variations of CYP epoxygenases are associated with the level of inflammatory markers. Also, in post-PCI population consisting of 1128 on-clopidogrel or on-prasugrel patients, CRP levels were observed to be regulated with a significant interaction between smoking and CYP2C19 polymorphisms; this effect was independent to the level of platelet aggregation. Additionally, in a large population of 1000 on-clopidogrel patients, whether there is a potential interaction between clopidogrel and calcium channel blockers. Collectively, we demonstrated in this thesis that inflammatory markers might be alternative tools for the prediction of response to thienopyridines
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Development of droplet-based microfluidic tools for toxicology and cancer research / Systèmes microfluidiques de crillage à haut débit en microgouttelettes pour la toxicologie et la recherche sur cancerLu, Heng 08 July 2016 (has links)
Ce projet de thèse portait sur le développement d’outils microfluidiques pour la toxicologie et la recherche contre le cancer. En permettant l’analyse simultanée d’un très grand nombre de réactions biologiques ou chimiques réalisés dans des compartiments indépendants (ie. gouttelettes), la microfluidique de gouttes offre une sensibilité de détection et une précision sans précédent pour l’analyse de molécules biologiques, telles que l’ADN ou les Anticorps, en comparaison des expériences réalisées conventionnellement en tubes ou en microplaques (essais en « bulk » ou volume). Ce format permet également de réaliser des expériences à très haut débit et est particulièrement pertinent pour la toxicologie, où des analyses robustes de l’effet des médicaments sont nécessaires. De même, ces procédures sont également très adaptées à l’analyse de cellules uniques pour le séquençage ADN ou ARN et l’épigénomique. Tout cela fait de la microfluidique en goutte un outil puissant pour la toxicologie et la recherche sur le cancer. En premier temps, une méthode du comptage précise des cellules encapsulée dans des microgouttelettes, nommée « hémocytométrie microfluidique », a été développée. Un nouvel algorithme de comptage a été proposé. Des cellules bactériennes (Escherichia Coli) et des cellules de 2 lignées humaines différentes (HL60 and H1975) ont été testées. Le nombre de chaque type de cellules a été déterminé avec une haute corrélation entre la théorie (basée sur la distribution de Poisson) et les résultats expérimentaux. Avec ces résultats robustes, un protocole de microfluidique en goutte a été mis en place pour interroger la viabilité cellulaire et la prolifération des 2 lignées humaines. Ces résultats sont en concordance avec ceux de la littérature. Pour la toxicologie, 3 différents modèles, y compris des microsomes (extrait de cellules d’insectes infectées par un baculovirus exprimant le cytochrome P450 3A4 humain, CYP3A4), HepG2-CYP3A4 (modifiée génétiquement pour exprimer le gène CYP3A4 humain), et HepaRG, une lignée hépatique, ont été évaluées pour l’activité enzymatique du CYP3A4, une enzyme largement utilisée en routine pour le criblage de médicament candidat. Les microsomes ont permis de développer un essai fluorogénique permettant de mesurer l’inhibition du CYP3A4. Cependant, ni l’utilisation des microsomes ni des cellules HepG2 exprimant CYP3A4 n’a donné de résultats satisfaisants en microgouttelettes. L’utilisation des cellules HepaRG, une lignée cellulaire qui conserve la majorité de l’expression des cytochromes P450 et des récepteurs nucléaires nécessaire à leur expression, a montré des résultats encourageant à la fois sur les tests de mesure de l’activité enzymatique et d’analyse de l’induction du CYP3A4. Pour la recherche sur le cancer, 4 essais originaux de PCR digitale en gouttes ont été mis en place pour la détection et la quantification de mutations (NRAS, DNMT3A, SF3B1 and JAK2) importante pour les syndromes myélodysplasiques, un groupe hétérogène de maladies touchant les cellules souches hématopoïétiques caractérisées par une hématopoïèse inefficace et des cytopénies périphériques. Finalement, un essai de PCR sur cellule unique encapsulées au sein de billes agarose a été proposé. / This thesis project consists in developing droplet-based microfluidic tools for toxicology and cancer research. Owing to its large numbers of discretized volumes, sensitivity of detection of droplet-based microfluidics for biological molecules such as DNA and antibody is much higher than bulk assays. This high throughput format is particularly suitable for experiments where a robust dose-response curve is needed, as well as for single cell analysis with applications in genomic or sequencing and epigenetics. All above makes droplet-based microfluidics a powerful tool for toxicology and cancer research. In a first part of the work, an accurate cell counting method, named “microfluidics hemocytometry”, has been developed. A new counting algorithm was proposed to count the cells within each droplet. Escherichia Coli and two different human cell lines (HL60 and H1975) were used to validate our strategy. The number of each type of cells in droplets was determined with a high consistency between theory (Poisson distribution) and experimental results. With these robust results, a droplet-based microfluidic protocol has then been established to inquiry both cell viability and proliferation for the two human cell lines. The results are in good agreement with the one of the literature. For the toxicology, 3 different biological models, including microsomes (extracted from baculovirus-infected insect cell expressing human CYP3A4), HepG2-CYP3A4 (genetically modified to express the human CYP3A4 gene) and HepaRG liver cells lines were evaluated for enzymatic activity of cytochromes P450 (CYP3A4), a routinely used enzyme for drug candidate screening. Microsome-based assays were used to validate a fluorogenic inhibition assay. However neither microsome-based assay nor the assay using CYP3A4 expressing HepG2 gave satisfying results in droplet-based format. However, HepaRG cells, a hepatic function-conserved cell line with most cytochrome and related nuclear receptors, demonstrated high relevance both for enzymatic activity testing and CYP3A4 expression induction study. For cancer research, 4 different picoliter droplet-based PCR assays were developed for the detection and quantification of mutations (NRAS, DNMT3A, SF3B1 and JAK2) present in Myelodysplastic syndromes, a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Furthermore, a single cell multistep PCR assay using encapsulation of target DNA in agarose droplets was proposed.
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Régulation fonctionnelle de l’épididyme d’un rongeur déserticole, Psammomys obesus, CRETZSCHMAR, 1828 / Functional Regulation of Epididymis of Sand Rat Psammomys obesus, CRETZSCHMAR, 1828Menad, Rafik 17 February 2015 (has links)
Afin de mettre en évidence les principaux éléments de la voie androgénique et œstrogénique dans l’épididyme du rat des sables adulte, capturé dans la région de Beni Abbès, en Algérie, l’aromatase, l’œstradiol, les récepteurs des androgènes (RA) et des œstrogènes (REα, REβ, GPR30) ont été recherchés chez des animaux en saison d’activité, en saison de repos sexuel, chez des animaux castrés, castrés puis traités par la testostérone et chez des animaux ayant subi la ligature des canaux efférents. En saison d’activité, les RA sont ubiquitaires, l’aromatase est cytoplasmique par contre l’œstradiol est nucléaire et cytoplasmique. Les REα et le GPR30 sont principalement dans le cytoplasme apical par contre les REβ sont nucléaires. En saison de repos sexuel, les RA, l’aromatase, l’œstradiol, les REα et le GPR30 persistent, cependant, les REβ subissent une translocation cytoplasmique. Chez les animaux castrés, les RA, l’aromatase et l’œstradiol sont réduits par contre les REα persistent avec une faible intensité. Le GPR30 est cytoplasmique et nucléaire. Chez les animaux castrés puis traités, les RA, l’aromatase, l’œstradiol, les REα, les REβ et le GPR30 sont restaurés. Chez les animaux ligaturés, le RA est faiblement conservé uniquement dans l’épididyme proximal. L’aromatase et l’œstradiol sont conservés. Le signal des REα, des REβ et du GPR30 est fortement exprimé dans le noyau et le cytoplasme dans l’épididyme proximal par contre il est fortement exprimé uniquement pour les REα dans l’épididyme distal. Par western blot, les RA, REα, REβ et GPR30 sont de 122, 64, 55 et 55 kDa respectivement. / In order to highlight the main elements of androgen and estrogen pathway in the epididymis of sand rat, captured in Beni Abbès area, in Algeria, androgen receptor (AR), aromatase, estradiol, estrogen receptors (ERα, ERβ and GPR30) were explored in breeding season, in resting season and in animals underwent castration, castration then testosterone treatment and ligation of efferent ducts. In breeding season, AR has a ubiquitous distribution, aromatase is exclusively cytoplasmic and estradiol is nuclear and cytoplasmic. The ERα and GPR30 were distributed with a high intensity in the apical cytoplasm contrarily to ERβ which were nuclear. In resting season, AR, aromatase, estradiol, ERα persist with lower staining. However, ERβ undergo cytoplasmic translocation and GPR30 persist in cytoplasm. In castrated animals, AR, aromatase and estradiol are reduced. ERα persist with low intensity in the apical cytoplasm. GPR30 is distributed in the cytoplasm and the nucleus. In castrated then treated animals, AR is restored; aromatase and estradiol reappear with a cytoplasmic localization for aromatase, nuclear and apical for ERα. ERβ and GPR30 are restored and have a cytoplasmic localization. In ligatured, RA is preserved in the caput, aromatase and estradiol persist caput and cauda. The signal of ERα, ERβ and GPR30 is highly expressed in the nucleus and cytoplasm of caput epididymis and highly expressed of ERα exclusively in cauda. By Western blot, RA, ERα, ERβ and GPR30 are found with molecular weights of 122, 64, 55 and 55 kDa respectively.
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The extracellular peroxygenase of the agaric fungus Agrocybe aegerita: catalytic properties and physiological background with particular emphasis on ether cleavage / Die extrazelluläre Peroxygenase des Lammellenpilzes Agrocybe aegerita: Katalytische Eigenschaften und physiologischer Hintergrund unter besonderer Berücksichtigung der EtherspaltungKinne, Matthias 11 November 2010 (has links) (PDF)
Litter-decay fungi have recently been shown to secrete heme-thiolate peroxygenases that oxidize various organic chemicals, but little is known about the physiological role or the mechanism of these enzymes. The aromatic peroxygenase of Agrocybe aegerita (AaeAPO) was purified and catalytically characterized. An overall reaction mechanism was proposed. The results show that AaeAPO catalyzed diverse H2O2-dependent monooxygenations (two-electron oxidations) including (a) the cleavage of aliphatic and aromatic ethers, (b) the regio- and enantioselective hydroxylation of aromatic compounds, (c) the stepwise oxygenation of benzylic compounds, (d) the N-dealkylation of secondary amines and (e) the dehalogenation of halogenated aliphatic compounds as well as typical peroxidase reactions (suggested to involve one-electron oxidation) such as (f) oxidation and polymerization of phenols and (g) halogenations. The enzyme failed to oxidize polymers such as polyethylene glycol (PEG).
Mechanistic studies with several model substrates provided information about the reaction cycle of AaeAPO: (1) stoichiometry of tetrahydrofuran cleavage showed that the reaction was a two-electron oxidation that generated one aldehyde group and one alcohol group, yielding the ring-opened product 4-hydroxybutanal, (2) steady-state kinetics results with methyl 3,4-dimethoxybenzyl ether, which was oxidized to 3,4-dimethoxybenzaldehyde, gave parallel double reciprocal plots suggestive of a ping-pong mechanism, (3) the cleavage of methyl 4-nitrobenzyl ether, the hydroxylation of aromatics such as diclofenac and nitrophenol and the oxygenation of benzylic compounds, resulted in incorporation of 18O into the reaction product in the presence of H218O2, and (4) the demethylation of 1-methoxy-4-trideuteromethoxybenzene showed an distinct observed intramolecular deuterium isotope effect. These results support a mechanism similar to that envisaged for the peroxygenase activity of P450s in which the enzyme heme is oxidized by H2O2 to give an iron species that carries one of the peroxide oxygen. This intermediate then abstracts a hydrogen from the substrate, which is followed by rebound of an •OH equivalent to produce the monooxygenated reaction product (hydrogen abstraction and oxygen rebound mechanism).
AaeAPO may accordingly have a role in the biodegradation of natural and anthropogenic low molecular weight compounds in soils and plant litter. Moreover, the results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites and herbicide precursors. / Die Peroxygenase des Südlichen Ackerling (Agrocybe aegerita, AaeAPO) wurde gereinigt, ihr Katalysepotential ermittelt und ein allgemeiner Reaktionsmechanismus postuliert. Die AaeAPO katalysiert sowohl H2O2-abhängige Monooxygenierungen (Zwei-Elektron Oxidationen) wie (a) die Spaltung aliphatischer und aromatischer Ether, (b) die regio- und enantioselektive Hydroxylierung von Aromaten, (c) die schrittweise Monooxygenierung von Toluolderivaten, (d) die N-Dealkylierung sekundärer Amine und (e) die Dehalogenierung chlorierter Aliphaten als auch typische Reaktionen bekannter Peroxidasen (vermutlich Ein-Elektron-Oxidation) unter anderem (f) die Oxidation/ Polymerisierung von Phenolen und (g) die Halogenierung von Aromaten. Polymere Verbindungen wie Polyethylenglycol (PEG) werden nicht oxidiert.
Mechanistische Untersuchungen zur Etherspaltung am Beispiel der AaeAPO haben Einblick in den generellen Reaktionsmechanismus dieses neuen Enzymtyps ermöglicht: (1) die Stöchiometrie der Spaltung von Tetrahydrofuran entspricht der einer zwei-Elektron-Oxidation, (2) die Spaltung von Methyl-3,4-Dimethoxybenzylether zu 4-Dimethoxybenzaldehyd und Methanol ergaben parallele Verläufe für die ermittelten Ausgleichsgeraden in der doppelt reziproken Darstellung, was einem „Ping-Pong“-Reaktionsmechanismus entspricht (3) die Monooxygenierungen haben stets den Einbau eines aus dem Peroxid (H2O2) stammenden Sauerstoffatoms in das Produkt zur Folge, (4) die O-Dealkylierung von 1-Methoxy-4-Trideuterummethoxybenzol zeigt einen ausgeprägten Deuterium Isotopen Effekt, was auf die primäre Abspaltung eines Wasserstoffatoms vom Substratmolekül hindeutet. Demnach verläuft die Peroxygenase-katalysierte Monooxygenierung über Wasserstoffabstraktion und eine unmittelbar anschließende Sauerstoffrückbindung (hydrogen abstraction - oxygen rebound mechanism). Diese Reaktionsabfolge ähnelt dem sogenannten peroxide "shunt" pathway, der von einer Reihe Cytochrom-P450-abhängiger Monooxygenasen her bekannt ist.
Die physiologische Funktion der AaeAPO besteht möglicherweise in der extrazellulären Transformation und Detoxifikation niedermolekularer Pflanzeninhaltsstoffe, mikrobieller Metabolite und anthropogener Xenobiotika. Aufgrund der Stabilität und Unabhängigkeit der AaeAPO von teuren Kofaktoren ergeben sich vielversprechende biotechnologische Möglichkeiten zum Einsatz isolierter Biokatalysatoren in selektiven (bio)chemischen Synthesen monooxygenierter Metabolite.
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