Fungal biotransformation of morphine alkaloids

The purpose of the study was to determine the ability of certain fungi to biotransform morphine alkaloids into medicinally relevant intermediates. Fungal strains screened for their ability to affect biotransformation of morphine alkaloids include Cunninghamella echinulata, Helicostylum pirijorme, Pycnoporus sanguinea, Pycnoporus cinnabarina, Curvularia lunata and Sporotrichum sulfurescens. The research demonstrated that Cunninghamella echinulata N-demethylated thebaine, hydrocodone, codeine, oripavine and oxycodone into corresponding nor-compounds in varying yields. The study further focused on the characterization of the enzyme responsible for the biotransformation of thebaine into northebaine by Cunninghamella echinulata. The study clearly showed that incubation of the fungal culture with thebaine over a period of 48 hours was required to activate the biotransformation process. The biotransformation studies with [14C] labeled thebaine showed that Ndemethylation by Cunningham ella echinulata does not involve O-demethylation followed by methyl group transfer as suggested in previous studies.

Dynamic epigenetic changes in immune responses to infection in human dendritic cells

La méthylation de l'ADN est une marque épigénétique importante chez les mammifères. Malgré le fait que la méthylation de la cytosine en 5' (5mC) soit reconnue comme une modification épigénétique stable, il devient de plus en plus reconnu qu'elle soit un processus plus dynamique impliquant des voies de méthylation et de déméthylation actives. La dynamique de la méthylation de l'ADN est désormais bien caractérisée dans le développement et dans le fonctionnement cellulaire des mammifères. Très peu est cependant connu concernant les implications régulatrices dans les réponses immunitaires. Pour se faire, nous avons effectué des analyses du niveau de transcription des gènes ainsi que du profilage épigénétique de cellules dendritiques (DCs) humaines. Ceux-ci ont été faits avant et après infection par le pathogène Mycobacterium tuberculosis (MTB). Nos résultats fournissent le premier portrait génomique du remodelage épigénétique survenant dans les DCs en réponse à une infection bactérienne. Nous avons constaté que les changements dans la méthylation de l'ADN sont omniprésents, identifiant 3,926 régions différentiellement méthylées lors des infections par MTB (MTB-RDMs). Les MTB-RDMs montrent un chevauchement frappant avec les régions génomiques marquées par les histones associées avec des régions amplificatrices. De plus, nos analyses ont révélées que les MTB-RDMs sont activement liées par des facteurs de transcription associés à l'immunité avant même d'être infecté par MTB, suggérant ces domaines comme étant des éléments d'activation dans un état de dormance. Nos données suggèrent que les changements actifs dans la méthylation jouent un rôle essentiel pour contrôler la réponse cellulaire des DCs à l'infection bactérienne.

Effets de la reprogrammation sur le gène empreinté H19 chez les équins

Lors de la fécondation, le génome subit des transformations épigénétiques qui vont guider le développement et le phénotype de l’embryon. L'avènement des techniques de reprogrammation cellulaire, permettant la dédifférenciation d'une cellule somatique adulte, ouvre la porte à de nouvelles thérapies régénératives. Par exemple, les procédures de transfert nucléaire de cellules somatique (SCNT) ainsi que la pluripotence par induction (IP) visent à reprogrammer une cellule somatique adulte différentiée à un état pluripotent similaire à celui trouvé durant la fécondation chez l'embryon sans en impacter l'expression génique vitale au fonctionnement cellulaire. Cependant, la reprogrammation partielle est souvent associée à une mauvaise méthylation de séquences géniques responsables de la régulation des empreintes géniques. Ces gènes, étudiés chez la souris, le bovin et l'humain, sont exprimés de manière monoallélique, parent spécifique et sont vitaux pour le développement embryonnaire. Ainsi, nous avons voulu définir le statut épigénétique du gène empreinté H19 chez l'équin, autant chez le gamètes que les embryons dérivés de manière in vivo, SCNT ainsi que les cellules pluripotentes induites (iPSC). Une région contrôle empreinté (ICR) riche en îlots CpG a été observée en amont du promoteur. Couplé avec une analyse de transcrit parent spécifique du gène H19, nous avons confirmé que l'empreinte du gène H19 suit le modèle insulaire décrit chez les autres mammifères étudiés et résiste à la reprogrammation induite par SCNT ou IP. La déméthylation partielle de l'ICR observée chez certains échantillons reprogrammés n'était pas suffisante pour induire une expression biallélique, suggérant un contrôle des empreintes chez les équins durant la reprogrammation.

Role of histone methylation in the regulation of COX-2, iNOS, and mPGES-1 gene expression in human chondrocytes: Implication for Osteoarthritis

L'arthrose (OA) est une maladie articulaire dégénérative, classée comme la forme la plus fréquente au monde. Elle est caractérisée par la dégénérescence du cartilage articulaire, l’inflammation de la membrane synoviale, et le remodelage de l’os sous-chondral. Ces changements structurels et fonctionnels sont dues à de nombreux facteurs. Les cytokines, les prostaglandines (PG), et les espèces réactives de l'oxygène sont les principaux médiateurs impliqués dans la pathophysiologie de l'OA. L'interleukine-1β (IL-1β) est une cytokine pro-inflammatoire majeure qui joue un rôle crucial dans l'OA. L'IL-1β induit l'expression de la cyclooxygénase-2 (COX-2), la microsomale prostaglandine E synthase-1 (mPGES-1), la synthase inductible de l'oxyde nitrique (iNOS), ainsi que leurs produits la prostaglandine E2 (PGE2) et l'oxyde nitrique (NO). Ce sont des médiateurs essentiels de la réponse inflammatoire au cours de l'OA qui contribuent aux mécanismes des douleurs, de gonflement, et de destruction des tissus articulaires. Les modifications épigénétiques jouent un rôle très important dans la régulation de l’expression de ces gènes pro-inflammatoires. Parmi ces modifications, la méthylation/ déméthylation des histones joue un rôle critique dans la régulation des gènes. La méthylation/ déméthylation des histones est médiée par deux types d'enzymes: les histones méthyltransférases (HMT) et les histones déméthylases (HDM) qui favorisent l’activation et/ou la répression de la transcription. Il est donc nécessaire de comprendre les mécanismes moléculaires qui contrôlent l’expression des gènes de la COX-2, la mPGES-1, et l’iNOS. L'objectif de cette étude est de déterminer si la méthylation/déméthylation des histones contribute à la régulation de l’expression des gènes COX-2, mPGES-1, et iNOS dans des chondrocytes OA humains induits par l'IL-1β. Nous avons montré que la méthylation de la lysine K4 de l'histone H3 (H3K4) par SET-1A contribue à l’activation des gènes COX-2 et iNOS dans les chondrocytes humains OA induite par l'IL-1β. Nous avons également montré que la lysine K9 de l’histone H3 (H3K9) est déméthylée par LSD1, et que cette déméthylation contribue à l’expression de la mPGES-1 induite par IL-1β dans les chondrocytes humains OA. Nous avons aussi trouvé que les niveaux d'expression des enzymes SET-1A et LSD1 sont élevés au niveau du cartilage OA. Nos résultats montrent, pour la première fois, l'implication de la méthylation/ déméthylation des histones dans la régulation de l’expression des gènes COX-2, mPGES-1, et iNOS. Ces données suggèrent que ces mécanismes pourraient être une cible potentielle pour une intervention pharmacologique dans le traitement de la physiopathologie de l'OA.

Characterization of DnmA, the Dnmt2 homolog in Dictyostelium discoideum

Eukaryotic DNA m5C methyltransferases (MTases) play a major role in many epigenetic regulatory processes like genomic imprinting, X-chromosome inactivation, silencing of transposons and gene expression. Members of the two DNA m5C MTase families, Dnmt1 and Dnmt3, are relatively well studied and many details of their biological functions, biochemical properties as well as interaction partners are known. In contrast, the biological functions of the highly conserved Dnmt2 family, which appear to have non-canonical dual substrate specificity, remain enigmatic despite the efforts of many researchers. The genome of the social amoeba Dictyostelium encodes Dnmt2-homolog, the DnmA, as the only DNA m5C MTase which allowed us to study Dnmt2 function in this organism without interference by the other enzymes. The dnmA gene can be easily disrupted but the knock-out clones did not show obvious phenotypes under normal lab conditions, suggesting that the function of DnmA is not vital for the organism. It appears that the dnmA gene has a low expression profile during vegetative growth and is only 5-fold upregulated during development. Fluorescence microscopy indicated that DnmA-GFP fusions were distributed between both the nucleus and cytoplasm with some enrichment in nuclei. Interestingly, the experiments showed specific dynamics of DnmA-GFP distribution during the cell cycle. The proteins colocalized with DNA in the interphase and were mainly removed from nuclei during mitosis. DnmA functions as an active DNA m5C MTase in vivo and is responsible for weak but detectable DNA methylation of several regions in the Dictyostelium genome. Nevertheless, gel retardation assays showed only slightly higher affinity of the enzyme to dsDNA compared to ssDNA and no specificity towards various sequence contexts, although weak but detectable specificity towards AT-rich sequences was observed. This could be due to intrinsic curvature of such sequences. Furthermore, DnmA did not show denaturant-resistant covalent complexes with dsDNA in vitro, although it could form covalent adducts with ssDNA. Low binding and methyltransfer activity in vitro suggest the necessity of additional factor in DnmA function. Nevertheless, no candidates could be identified in affinity purification experiments with different tagged DnmA fusions. In this respect, it should be noted that tagged DnmA fusion preparations from Dictyostelium showed somewhat higher activity in both covalent adduct formation and methylation assays than DnmA expressed in E.coli. Thus, the presence of co-purified factors cannot be excluded. The low efficiency of complex formation by the recombinant enzyme and the failure to define interacting proteins that could be required for DNA methylation in vivo, brought up the assumption that post-translational modifications could influence target recognition and enzymatic activity. Indeed, sites of phosphorylation, methylation and acetylation were identified within the target recognition domain (TRD) of DnmA by mass spectrometry. For phosphorylation, the combination of MS data and bioinformatic analysis revealed that some of the sites could well be targets for specific kinases in vivo. Preliminary 3D modeling of DnmA protein based on homology with hDNMT2 allowed us to show that several identified phosphorylation sites located on the surface of the molecule, where they would be available for kinases. The presence of modifications almost solely within the TRD domain of DnmA could potentially modulate the mode of its interaction with the target nucleic acids. DnmA was able to form denaturant-resistant covalent intermediates with several Dictyostelium tRNAs, using as a target C38 in the anticodon loop. The formation of complexes not always correlated with the data from methylation assays, and seemed to be dependent on both sequence and structure of the tRNA substrate. The pattern, previously suggested by the Helm group for optimal methyltransferase activity of hDNMT2, appeared to contribute significantly in the formation of covalent adducts but was not the only feature of the substrate required for DnmA and hDNMT2 functions. Both enzymes required Mg2+ to form covalent complexes, which indicated that the specific structure of the target tRNA was indispensable. The dynamics of covalent adduct accumulation was different for DnmA and different tRNAs. Interestingly, the profiles of covalent adduct accumulation for different tRNAs were somewhat similar for DnmA and hDNMT2 enzymes. According to the proposed catalytic mechanism for DNA m5C MTases, the observed denaturant-resistant complexes corresponded to covalent enamine intermediates. The apparent discrepancies in the data from covalent complex formation and methylation assays may be interpreted by the possibility of alternative pathways of the catalytic mechanism, leading not to methylation but to exchange or demethylation reactions. The reversibility of enamine intermediate formation should also be considered. Curiously, native gel retardation assays showed no or little difference in binding affinities of DnmA to different RNA substrates and thus the absence of specificity in the initial enzyme binding. The meaning of the tRNA methylation as well as identification of novel RNA substrates in vivo should be the aim of further experiments.

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Palythine-threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata

Using a high-resolution reverse-phase liquid chromatography method we found that the tissues of the hermatypic coral Pocillopora capitato (collected in Santiago Bay, Mexico) contain a high diversity of primary and secondary mycosporine-like amino acids (MAAs) typical of some reef-building coral species: mycosporine-glycine, shinorine, porphyra-334, mycosporine-methylamine-serine, mycosporine-methylamine-threonine, palythine-serine, palythine and one additional novel predominant MAA, with an absorbance maximum of 320 nm. Here we document the isolation and characterization of this novel MAA from the coral A capitata. Using low multi-stage mass analyses of deuterated and non deuterated compounds, high-resolution mass analyses (Time of Flight, TOF) and other techniques, this novel compound was characterized as palythine-threonine. Palythine-threonine was also present in high concentrations in the corals Pocillopora eydouxi and Stylophora pistillata indicating a wider distribution of this MAA among reef-building corals. From structural considerations we suggest that palythine-threonine is formed by decarboxylation of porphyra-334 followed by demethylation of mycosporine-methylamine-threonine. (C) 2008 Elsevier B.V. All rights reserved.

Metabolism of (-)-grandisin from Piper solmsianum in Coleoptera and Lepidoptera species

The biotransformation of the major Piper solmsianum leaf phenylpropanoids, such as the tetrahydrofuran lignan grandisin and derivatives was investigated in the beetle Naupactus bipes as well as in the caterpillars Heraclides hectorides and Quadrus u-lucida. Analysis of fecal material indicated that metabolism occurred mainly through mono- and di-O-demethylation at para positions of 3,4,5-trimethoxyphenyl rings of tetrahydrofuran lignans during digestion by these insects. Additionally, 3-hydroxy-4,5-dimethoxycinnamyl and 3,4,5-trimethoxycinnamyl alcohols were identified in fecal extracts of N. bipes. (C) 2008 Elsevier Ltd. All rights reserved.

Evolution of a contagious cancer: epigenetic variation in devil facial tumour disease

The emergence of Devil Facial Tumour Disease (DFTD), a highly contagious cancer, is driving Tasmanian devils (Sarcophilus harrisii) to extinction. The cancer is a genetically and chromosomally stable clonal cell line which is transmitted by biting during social interactions. In the present study, we explore the Devil Facial Tumour (DFT) epigenome and the genes involved in DNA methylation homeostasis. We show that tumour cells have similar levels of methylation to peripheral nerves, the tissue from which DFTD originated. We did not observe any strain or region-specific epimutations. However, we revealed a significant increase in hypomethylation in DFT samples over time (p < 0.0001). We propose that loss of methylation is not because of a maintenance deficiency, as an upregulation of DNA methyltransferase 1 gene was observed in tumours compared with nerves (p < 0.005). Instead, we believe that loss of methylation is owing to active demethylation, supported by the temporal increase in MBD2 and MBD4 (p < 0.001). The implications of these changes on disease phenotypes need to be explored. Our work shows that DFTD should not be treated as a static entity, but rather as an evolving parasite with epigenetic plasticity. Understanding the role of epimutations in the evolution of this parasitic cancer will provide unique insights into the role of epigenetic plasticity in cancer evolution and progression in traditional cancers that arise and die with their hosts.

The effect of pH on horseradish peroxidase-catalyzed oxidation of melatonin: Production of N1-acetyl-N2-formyl-5- methoxykynuramine versus radical-mediated degradation

There is a growing body of evidence that melatonin and its oxidation product, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5, 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin, a dimer of 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. On the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle. © 2007 The Authors.

Avaliação antropometrica e bioquímica em pacientes renais crônicos e a ação da suplementação de ácido fólico na homocisteína, lipídeos, albumina e proteína C reativa

Pós-graduação em Alimentos e Nutrição - FCFAR

Qualidade de frutos de tomate ‘giuliana’ tratados com produtos de efeitos fisiológicos

The present work evaluated the effect of strobilurins, boscalid, plant growth regulators and vegetal extracts on the physico-chemical quality of tomato fruits (Solanum lycopersicum L.), hybrid Giuliana. The fruits from each treatment were selected and separated in 4 repetitions: control, pyraclostrobin, boscalid, pyraclostrobin + boscalid, IBA + GA(3) + kinetin, GA(4+7) + benzylaminopurine and vegetal extract. The first application was carried out at 30 days after transplant and the following at every 15 days. The evaluations were: weight loss, titratable acidity (TA), soluble solids (SS), SS/TA relation, pH, ascorbic acid content, texture, total soluble sugars (TSS), activity of pectin methyl esterase (PME) and polygalacturonase (PG). The SS content, responsible for fruit flavor, varied accordingly to the treatment, being highest for GA(4+7) + benzylaminopurine and lowest for the control. The same occurred regarding the SS/AT relation. The fruits were kept on the shelf for 9 days, at room temperature, being that at the end of this period several treatments still presented fruit in optimal consumption conditions, notably the boscalid treatment, which presented lowest weight loss, followed by pyraclostrobin. Therefore, it can be concluded that the application of the treatments did not modify the values for pH, AT and AST of the fruits. The highest PME activity was observed for the treatments with boscalid and the mixture of boscalid and pyraclostrobin, while the lowest PG activity occurred in the control and the pyraclostrobin treatment, indicating that some products accelerated the process of demethylation of pectins by PME, facilitating the action of PG.

Vanadato de bismuto sintetizado por combustão em solução na presença de diferentes combustíveis: síntese, caracterização e estudo da atividade fotocatalítica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Distribuição generalizada da resistência a fungicidas triazóis e evolução do gene cyp51A em populações de Pyricularia oryzae da brusone do trigo no Brasil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)