Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By using calcium flux assay in a flow-cytometer, we found that trypsin-treated keratinocytes showed an increase in intracellular calcium release. To test whether PAR2 mediates the escape response to trypsin, we treated larvae with a PAR2 antagonist and showed that the trypsin-initiated escape response was abrogated. Furthermore, par2a mutants with knockdown of par2a by the piggyback knockdown method failed to respond to trypsin. Trypsin treatment of adult fish led to an approximately 2-fold increase in brain c-fos mRNA levels 45 mins after trypsin treatment, suggesting that trypsin signals may have reached the brain, probably via a spinothalamic pathway. Taken together, our results reveal a novel trypsin-initiated escape response in fish. These studies should enhance our understanding of fish communication in general and alarm behavior in particular. Furthermore, since pain receptors in other animals are also PAR2, our finding may be useful in exploring pathways of pain reception.

No description available.

No description available.

A fatty acid amide is precisely as the name suggests: A fatty acid (CHn-COOH), in which the hydroxyl group of the carboxylic acid is displaced by an amine functional group from a biogenic amine (R-NH2), ultimately forming an amide bond. Furthermore, these fatty acid amides can be composed of a variety of different acyl chain lengths donated by the fatty acid and a myriad of different biogenic amines. Thus, these molecules can be subdivided in a number of different ways including the separation of short chain (acetyl to heptanoyl) and long chain (palmitoyl to arachidonoyl) and also based off the biogenic amine type. The long chain fatty acid amides quickly gained the interest of the scientific community through the discovery of anandamide (N-arachidonoylethanolamide), which was found to be the endogenous ligand for the cannabinoid receptor-1 (CB1) found in the mammalian brain. This particular neural molecule is an N-acylethanolamide, which is one specific classification of long chain fatty acid amide. However, there exist other types of long chain fatty acid amides including the N-acylglycines, primary fatty acid amides (PFAMs) and N-acylarylalkylamides. Yet, despite the type of fatty acid amide, it has been shown many of these types of molecules are synthesized using a type of N-acyltransferase. These N-acyltransferases are believed to be members of the GCN5-related superfamily of N-acyltransferases (GNAT), which share the feature of being able to accept acyl-CoA thioester substrates. This dissertation will discuss and demonstrate the extraction of all types of the aforementioned classifications of long chain fatty acid amides but will have a particular focus on the N-acylarylalkylamides. Elucidating more about the biosynthetic pathways and metabolic routes of the long chain fatty acid amides could lead to the development of potential therapeutics and pest control agents. We have determined Drosophila melanogaster arylalkylamine N-acyltransferase like 2 is responsible for the in vivo biosynthesis of N-acyldopamines. We have also demonstrated Bombyx mori is another suitable model systems for the study of long chain fatty acid amides, as three insect arylalkylamine N-acyltrasnferase from Bombyx mori (Bm-iAANAT) were found to share some homology in primary sequence (25-29%) to AAANTL2 in Drosophila melanogaster. We show herein that one of these enzymes is able to catalyze the formation of long chain N-acylarylalkylamides in vivo. The change in the transcription of these enzymes was tracked to try and understand if these enzymes serve a focused purpose in the physiological development of the insect. If it is found one of these Bm-iAANAT are crucial for growth, it may elucidate a general function of the enzyme, which may be able to inhibit growth of specific insects that are known pests, while not targeting endangered insects like Apis melliferra (honey bee). Understanding this would help in the eventual creation of targeted insecticides on specific insect pests Furthermore, a novel panel of fatty acid amides was characterized and quantified in extracts from this organism via LC-QToF-MS, ultimately showing it is very possible the Bm-iAANATs are performing this catalysis in vivo.

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2012-08-13T05:08:16Z No. of bitstreams: 1 ademir_j_martins_junior_ioc_bp_0028_2009.pdf: 3828128 bytes, checksum: 594e5415edb29a09e864f5395428b97c (MD5) / Made available in DSpace on 2012-08-13T05:08:16Z (GMT). No. of bitstreams: 1 ademir_j_martins_junior_ioc_bp_0028_2009.pdf: 3828128 bytes, checksum: 594e5415edb29a09e864f5395428b97c (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / No Brasil o controle das formas aladas do vetor de dengue, o mosquito Aedes aegypti, é feito com inseticidas da classe dos piretróides. Porém, apesar da recente utilização deste composto em escala nacional, várias populações do vetor já estão resistentes. O canal de sódio regulado por voltagem, no sistema nervoso do inseto, é a molécula-alvo de piretróides. Investigamos, em populações brasileiras de Ae. aegypti, a diversidade molecular em uma região deste gene (AaNaV) com o objetivo de identificar potenciais alterações relacionadas à resistência. Clonagem e sequenciamento da região genômica entre os exons 20 e 21 do AaNaV de indivíduos de cinco localidades distintas, confirmaram polimorfismo no íntron e em dois sítios do exon 20 que geram substituições sinônimas. De acordo com estas características, as sequências foram agrupadas em tipos A ou B. Observamos ainda mutações causando substituições de aminoácidos: Ile  Met no sítio 1011 e Val  Ile no sítio 1016, somente em sequências do tipo A. Indivíduos da cepa Rockefeller, referência de susceptibilidade, apresentaram apenas sequências do tipo B e os alelos selvagens nas duas posições, 1011 ou 1016. Tipagem molecular por PCR alelo-específica em indivíduos de 15 localidades revelou que a mutação Ile1011Met está disseminada por todo país, diferentemente da Val1016Ile, concentrada nas regiões mais ao centro. Nas cinco localidades onde os indivíduos avaliados foram divididos em resistentes e susceptíveis, o alelo mutante 1016Ile esteve significativamente mais presente nos resistentes, principalmente quando em homozigose, indicando o caráter recessivo da mutação para a resistência. Surpreendentemente, uma série de observações sugeriu a ocorrência de polimorfismo envolvendo uma duplicação gênica, de forma que o haplótipo duplicado estaria constituído de uma sequência do tipo B ligada à outra do tipo A com a mutação 1011Met. Corroborou esta hipótese a tipagem do sítio 1011 na prole de cruzamentos com genótipos determinados. Finalmente, comparação entre linhagens mantidas na presença ou na ausência de pressão de seleção com piretróide, em laboratório, sugeriu efeitos pleiotrópicos negativos da resistência em aspectos do desenvolvimento e da reprodução destes mosquitos / In Brazil, control of adults of the dengue vector, the mosquito Aedes aegypti, is performed with pyrethroid insecticides. However, despite the recent implementation of this compound in national scale, several populations of this vector are already resistant. The voltage gated sodium channel is the pyrethroid target site, in the insect nervous system. We investigated the molecular diversity of a particular region of this gene (AaNaV) in Ae. aegypti Brazilian populations in order to identify potential substitutions implicated with insecticide resistance. Cloning and sequencing of the genome region between the AaNaV exons 20 and 21 in individuals from five distinct localities, confirmed polymorphism in the intron and in two positions of exon 20, these latter related to synonymous substitutions. According to these characteristics sequences were grouped into types A or B. Two additional predictive substitutions were noted: Ile Met and Val  Ile, respectively, in 1011 and 1016 sites, both only in type A sequences. Individuals from the Rockefeller insecticide susceptible reference strain exhibited only type B sequences and the wild alleles on both 1011 and 1016 positions. Allelespecific PCR molecular typing of individuals from 15 localities showed that the Ile1011Met mutation is widespread throughout Brazil, whereas Val1016Ile is more proeminent toward the middle of the country. In five localities typing was performed separately in susceptible or resistant individuals; the mutant 1016Ile allele was significantly more present in the resistant ones, especially in homozygozity, suggesting the recessive character of this resistant mutation. Surprisingly, a series of observations suggested the occurrence of a gene duplication consisting of both type B and A sequences this last one with the 1011Met mutation. Typing of the 1011 site in the offspring of couples with specific genotypes corroborated this hypothesis. Finally, comparisons among lineages selected or not with pyrethroid suggested negative pleiotropic effects of resistance on development and reproduction aspects.

No description available.

No description available.

Le stress oxydant joue un rôle majeur dans le développement et l’évolution des maladies inflammatoires de l’intestin. Le corps humain est doté d’une panoplie d’enzymes antioxydantes ayant pour fonction de protéger l’intégrité cellulaire. De nouvelles enzymes au fort potentiel antioxydant, les paraoxonases (PON) 1, 2 et 3, ont récemment été identifiées tout au long du tube digestif, mais leurs rôles y restent inconnus. Les cellules intestinales Caco-2/15, qui ont la capacité de se différencier et d’acquérir les caractéristiques physiologiques de l'intestin grêle, ont été utilisées dans le présent travail pour étudier la régulation des PON. Les cellules ont été traitées avec différents effecteurs physiologiques (cytokines, LPS, stress oxydant) et pharmacologiques (fibrates, thiazolidinédiones) et l’expression des leurs gènes et protéines a été évaluée. Les résultats ont mis en lumière la modulation distincte de l’expression des PON par le stress oxydant et l’inflammation. Ceci suggère que chaque PON peut jouer un rôle différent au niveau intestinal et être impliquée dans le maintien de l’homéostasie. La régulation de l’expression des PON a également été largement explorée dans un article de revue. Pour définir le rôle de PON2, celle-ci étant potentiellement la plus importante pour l’homéostasie intestinale, les cellules Caco-2/15 ont été infectées à l’aide de lentivirus contenant des ARN d’interférence, ce qui a fortement réduit l’expression de PON2. En l’absence de PON2, les cellules Caco-2/15 étaient plus susceptibles face à un stress oxydant, la réponse inflammatoire était exacerbée et la perméabilité cellulaire paraissait altérée. Toutes ces composantes sont majeures dans le développement des maladies inflammatoires de l’intestin chez l’humain. De plus, des cellules Caco-2/15 de la PON2, ce qui a renforcé la force de la défense antioxydante cellulaire. Les résultats suggèrent que les PON jouent un rôle dans le maintien de l’homéostasie intestinale et pourraient être impliquées dans l’étiologie et la pathogenèse des maladies inflammatoires de l’intestin. / Oxidative stress is a major part of the pathogenesis of inflammatory bowel disease (IBD). The endogenous antioxidative defence is formed of multiple enzymes that have to protect cellular integrity. Paraoxonases (PON1, 2 and 3) are antioxidant enzymes that have recently been identified throughout the digestive tract, but their roles remain unclear in the intestine. Intestinal Caco-2/15 cells, which have the capacity to differentiate and exhibit the functionality of the small intestine, were used to study PON’s regulation. Cells were treated with various physiological effectors (cytokines, lipopolysaccharides, oxidative stress) and pharmacological molecules (fibrates, thiazolidinediones) and gene and protein expression were determined. Results obtained showed that PONs are distinctly modulated especially by inflammation and oxidative stress and suggests that they could play different roles in the maintenance of intestinal homeostasis. PONs regulation has also been the main topic of a review article. Our results and the literature pointed to PON2 as the most important PON for intestinal health. To better define PON2 functions in the intestine, PON2 expression was knocked-down using lentiviral infection and plasmids containing anti-PON2 shRNA. In the relative absence of PON2, Caco-2/15 cells were more susceptible towards induction of oxidative stress, the inflammatory response was exacerbated and cell permeability seemed altered. All of these components are major players involved in the development of human IBD. Moreover, Caco-2/15 cells were treated with purified PON2, which increased their antioxidative defence. All of these results suggest that PONs are implicated in the antioxidative and anti-inflammatory response in intestinal epithelial cells and makes them potentially important players for the aetiology and pathogenesis of IBD.

Les sites apuriniques/apyrimidiniques (AP) sont des sites de l’ADN hautement mutagène. Les dommages au niveau de ces sites peuvent survenir spontanément ou être induits par une variété d’agents. Chez l’humain, les sites AP sont réparés principalement par APE1, une enzyme de réparation de l’ADN qui fait partie de la voie de réparation par excision de base (BER). APE1 est une enzyme multifonctionnelle; c’est une AP endonucléase, 3’-diestérase et un facteur redox impliqué dans l’activation des facteurs de transcription. Récemment, il a été démontré qu’APE1 interagit avec l’enzyme glycolytique GAPDH. Cette interaction induit l’activation d’APE1 par réduction. En outre, la délétion du gène GAPDH sensibilise les cellules aux agents endommageant l’ADN, induit une augmentation de formation spontanée des sites AP et réduit la prolifération cellulaire. A partir de toutes ces données, il était donc intéressant d’étudier l’effet de la délétion de GAPDH sur la progression du cycle cellulaire, sur la distribution cellulaire d’APE1 et d’identifier la cystéine(s) d’APE1 cible(s) de la réduction par GAPDH. Nos travaux de recherche ont montré que la déficience en GAPDH cause un arrêt du cycle cellulaire en phase G1. Cet arrêt est probablement dû à l’accumulation des dommages engendrant un retard au cours duquel la cellule pourra réparer son ADN. De plus, nous avons observé des foci nucléaires dans les cellules déficientes en GAPDH qui peuvent représenter des agrégats d’APE1 sous sa forme oxydée ou bien des focis de la protéine inactive au niveau des lésions d’ADN. Nous avons utilisé la mutagénèse dirigée pour créer des mutants (Cys en Ala) des sept cystéines d’APE1 qui ont été cloné dans un vecteur d’expression dans les cellules de mammifères. Nous émettons l’hypothèse qu’au moins un mutant ou plus va être résistant à l’inactivation par oxydation puisque l’alanine ne peut pas s’engager dans la formation des ponts disulfures. Par conséquent, on anticipe que l’expression de ce mutant dans les cellules déficientes en GAPDH pourrait restaurer une distribution cellulaire normale de APE1, libérerait les cellules de l’arrêt en phase G1 et diminuerait la sensibilité aux agents endommageant l’ADN. En conclusion, il semble que GAPDH, en préservant l’activité d’APE1, joue un nouveau rôle pour maintenir l’intégrité génomique des cellules aussi bien dans les conditions normales qu’en réponse au stress oxydatif. / Apurinic/apyrimidinic (AP) sites are highly mutagenic DNA lesions occurring either spontaneously or by the action of DNA damaging agents. In human cells, AP sites are processed by the major DNA repair enzyme APE1 through the base excision repair (BER) pathway. APE1 is a multifunctional protein that has AP endonuclease/3’-diesterase activities in addition to its role as a redox factor in activating many transcription factor. Recently, it has been shown that APE1 interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an interaction that results in the activation of APE1 by reduction. Interestingly, depletion of GAPDH sensitized the cells to DNA damaging agents and induced an increase in spontaneous AP sites frequency. Moreover, cells knocked-down for GAPDH showed defects in proliferation. Here we set up to investigate the effects of GAPDH knockdown on cell cycle progression, APE1 subcellular localization and to identify the cysteine residue(s) of APE1, target(s) of GAPDH reduction. Our studies showed that GAPDH deficient cells arrested in G1 phase of the cell cycle. The defect in cell cycle progression is most probably due to accumulation of DNA damage which activates checkpoints leading to a delay in the cell cycle to allow DNA repair. Furthermore, in GAPDH deficient cells, APE1 formed nuclear foci-like structures that could represent aggregates of the oxidized form of APE1 or inactive APE1 foci on DNA lesions. Using site-directed mutagenesis, we created seven APE1 cysteine to alanine mutants which were cloned into a mammalian expression vector. We expect that at least one of these mutants is likely to resist the inactivation by oxidation as it cannot engage in disulfide bridge formation. Therefore, the expression of this mutant(s) in GAPDH knockdown cells is expected to restore a normal APE1 cellular distribution, rescue the cell cycle defects, and render the cells less sensitive to DNA damaging agents. In conclusion, our results show a new role of GAPDH in maintaining genomic stability under oxidative stress by maintaining APE1 in its functional state.

No description available.