The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

Endogenous brain compounds and xenobiotics, including the neurotoxins such as the amphetamine analogs 3,4-methylenedioxymethamphetamine (MDMA,Ecstasy), methamphetamine (METH, Speed) and methylenedioxyamphetamine (MDA, active metabolite of MDMA), may be bioactivated by prostaglandin H synthase (PHS) to free radicals that generate reactive oxygen species (ROS). In the absence of adequate antioxidant or repair mechanisms, ROS oxidize macromolecules such as DNA, protein and lipids, which can lead to toxicity. In vitro, we evaluated bioactivation using both purified ovine PHS-1 and cultured cells stably overexpressing either human PHS-1 or hPHS-2 isozymes. We found the neurotransmitter dopamine, its precursors and some metabolites, as well as METH and MDA, can be bioactivated by ovine and/or human PHS in an isozyme-dependent fashion that generates ROS, which oxidize DNA and protein and increase toxicity. This process is blocked by both the PHS inhibitor acetylsalicylic acid (ASA) and the ROS detoxifying enzyme catalase. Our data are the first to reveal isozyme-dependent bioactivation by PHS as a potential mechanism for enhanced susceptibility to both exogenous and endogenous neurotoxins, the latter of which may be particularly important in aging. METH-initiated ROS can also activate redox-sensitive transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf2), which is involved in the induction of an array of protective mechanisms in both adult and fetal brain. Using Nrf2 knockout mice, we showed Nrf2 has a novel neuroprotective role in METH-initiated oxidative stress, neurotoxicity and functional deficits in both fetal development and adulthood, especially with multiple exposures allowing time for the induction of neuroprotective mechanisms. Our studies are the first to show that Nrf2 afforded protection against both motor coordination deficits and olfactory deficits caused by METH in utero and in adults, suggesting that deficiencies in Nrf2 activation constitute a risk factor for ROS-mediated neurotoxicity in the fetus and adult.

Endogenous brain compounds and xenobiotics, including the neurotoxins such as the amphetamine analogs 3,4-methylenedioxymethamphetamine (MDMA,Ecstasy), methamphetamine (METH, Speed) and methylenedioxyamphetamine (MDA, active metabolite of MDMA), may be bioactivated by prostaglandin H synthase (PHS) to free radicals that generate reactive oxygen species (ROS). In the absence of adequate antioxidant or repair mechanisms, ROS oxidize macromolecules such as DNA, protein and lipids, which can lead to toxicity. In vitro, we evaluated bioactivation using both purified ovine PHS-1 and cultured cells stably overexpressing either human PHS-1 or hPHS-2 isozymes. We found the neurotransmitter dopamine, its precursors and some metabolites, as well as METH and MDA, can be bioactivated by ovine and/or human PHS in an isozyme-dependent fashion that generates ROS, which oxidize DNA and protein and increase toxicity. This process is blocked by both the PHS inhibitor acetylsalicylic acid (ASA) and the ROS detoxifying enzyme catalase. Our data are the first to reveal isozyme-dependent bioactivation by PHS as a potential mechanism for enhanced susceptibility to both exogenous and endogenous neurotoxins, the latter of which may be particularly important in aging. METH-initiated ROS can also activate redox-sensitive transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf2), which is involved in the induction of an array of protective mechanisms in both adult and fetal brain. Using Nrf2 knockout mice, we showed Nrf2 has a novel neuroprotective role in METH-initiated oxidative stress, neurotoxicity and functional deficits in both fetal development and adulthood, especially with multiple exposures allowing time for the induction of neuroprotective mechanisms. Our studies are the first to show that Nrf2 afforded protection against both motor coordination deficits and olfactory deficits caused by METH in utero and in adults, suggesting that deficiencies in Nrf2 activation constitute a risk factor for ROS-mediated neurotoxicity in the fetus and adult.

7,8-Linoleate diol synthase (7,8-LDS) of the take-all pathogen of wheat, Gaeumannomyces graminis, converts linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8-HPODE) by 8-dioxygenase activity (8-DOX), and further isomerizes the hydroperoxide to 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by hydroperoxide isomerase activity. Sequence alignment showed homology to prostaglandin H synthase (PGHS), and both enzymes share structural and catalytic properties. The 8-DOX of 7,8-LDS was successfully expressed in Pichia pastoris and in insect cells (Sf21). Site-directed mutagenesis confirmed His379 as the proximal heme ligand and Tyr376 as a residue, which forms a tyrosyl radical and initiates catalysis. Furthermore, mutagenesis suggested His203 could be the proposed distal histidine, and Tyr329 of catalytic relevance for substrate positioning at the active site. Aspergilli are ubiquitous environmental fungi. Some species, in particular Aspergillus fumigatus, are responsible for invasive aspergillosis, which is a life-threatening disease for immunocompromised patients. A. fumigatus and A. nidulans metabolized linoleic acid to 8R-HPODE, 10R-hydroperoxyoctadecadienoic acid (10R-HPODE), 5S,8R-dihydroxyoctadecadienoic acid, and 8R,11S-dihydroxyoctadecadienoic acid. When the genomes of certain Aspergilli strains were published, several species showed at least three homologous genes (ppoA, ppoB, ppoC- psi producing oxygenases) to 7,8-LDS and PGHS. Gene deletion identified PpoA as an enzyme with 8-DOX and 5,8-hydroperoxide isomerase activities, designated 5,8-LDS in homology to 7,8-LDS. In the same way, PpoC was identified as a 10-dioxygenase (10-DOX), which converts linoleic acid to 10R-HPODE. 10-DOX differs from LDS, since it dioxygenates linoleic acid at C-10, after hydrogen abstraction at C-8 and double bond migration. 10-DOX was cloned and expressed in insect cells. Leu384 and Val388 were found to be critical for dioxygenation at C-10. Mutation to the homologous residues of 5,8- and 7,8-LDS (Leu384Val, Val388Leu) increased oxygen insertion at C-8. LDS and 10-DOX are fusion proteins with a dioxygenase and a hydroperoxide isomerase (cytochrome P450) domain with a cysteine heme ligand. The P450 domain of 10-DOX lacked the crucial cysteine heme ligand and was without hydroperoxide isomerase activity. LDSs and 10-DOX are newly characterized heme containing fungal dioxygenases, with homology to PGHS of vertebrates. Their metabolites regulate reproduction, development, and act as signal molecules with the host after pathogen attack.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Epilepsy is one of the most common neurologic disorders. It has been suggested that seizures may be facilitaded by inflammation. PGE2 is one of the most important inflammatory mediators, and facilitates pentylenetetrazol (PTZ)-induced seizures by stimulating EP1 and EP3 receptors. However, up to the present moment, no study has investigated whether EP1 and EP3 receptors blocking attenuate seizures induced by convulsants other than PTZ. It is also unknown whether Na+,K+-ATPase activity alterations are involved in such an effect. Therefore, in the current study we investigated whether EP1 and EP3 ligands (agonists and antagonists) modulate PTZ- and kainic acid (KA)-induced seizures, and whether alterations in Na+,K+-ATPase activity mediate such a protective effect, in mice. EP1 and EP3 antagonists (ONO-8713 and ONO-AE3-240, respectively, 10 Og/kg, s.c.) attenuated PTZ (60 mg/kg, i.p.)- and KA (20 mg/kg, i.p.)-induced seizures. The respective agonists (ONO-DI-004 and ONO-AE-248, 10 Og/kg, s.c.) facilitated seizures in both acute models, and at noneffective doses, prevented the protective effects of the antagonists. Animals injected with PTZ presented decreased Na+,K+-ATPase activity in the cerebral cortex and hippocampus. On the other hand, animals injected with KA presented increased Na+,K+-ATPase activity in the same cerebral structures at the end of the experiment. These divergent findings suggest that alterations in Na+,K+-ATPase activity in both acute models depends on the convulsant agent used and make difficult to establish a relationship between Na+,K+-ATPase activity and seizure development. Moreover, EP1 and EP3 antagonists administration abolished Na+,K+- ATPase activity alterations induced by PTZ and KA, in such a way that these alterations seem to be related more to the presence of ictal phenomenon itself than to the seizure induction mechanisms. Notwithstanding, the currrent results clearly show that EP1 and EP3 receptors might constitute novel targets for anticonvulsants development, since EP1 and EP3 decreased seizures, regardless of the convulsant agent used. / A epilepsia é uma das disfunções neurológicas mais comuns. Tem sido sugerido que as crises epilépticas podem ser facilitadas pela ocorrência de inflamação. A PGE2 é um dos mediadores inflamatórios mais importantes que, agindo por meio dos receptores EP1 e EP3, facilita as convulsões induzidas por pentilenotetrazol (PTZ). Contudo, até a presente data, nenhum estudo investigou, de maneira sistêmica, se a ativação ou bloqueio de receptores EP1 e EP3 facilitam as convulsões induzidas por outros agentes; tampouco se alterações na atividade da Na+,K+-ATPase estão envolvidas nesse efeito. Assim, no presente estudo, investigamos se ligantes (agonistas e antagonistas) de receptores EP1 e EP3 modificam as crises induzidas por PTZ e ácido caínico (KA), e se tais efeitos estão associados a alterações na atividade da enzima Na+,K+-ATPase, em camundongos. Os antagonistas EP1 e EP3 (ONO-8713 e ONO-AE3-240, respectivamente, 10 Og/Kg, s.c.) atenuaram as convulsões induzidas por PTZ (60 mg/Kg, i.p.) e KA (20 mg/Kg). Os seus respectivos agonistas (ONO-DI-004 e ONO-AE-248 de 10 Og/Kg, s.c.) facilitaram as convulsões em ambos modelos agudos de crises epilépticas e, em doses não efetivas para gerar crises, preveniram os efeitos dos antagonistas. Os animais submetidos à administração de PTZ apresentaram, ao final do experimento, a atividade Na+,K+-ATPásica diminuída no córtex cerebral e hipocampo. Por outro lado, animais tratados com KA apresentaram um aumento na atividade Na+,K+-ATPásica nestas mesmas estruturas, que se correlacionou positivamente com a vigência de status epilepticus no momento do sacrifício. Os achados divergentes no que diz respeito à alteração da atividade da Na+,K+-ATPase nos dois modelos de crises agudas sugere que tais alterações estejam relacionadas ao tipo de agente convulsivante utilizado, e dificultam estabelecer, de forma inequívoca, uma relação entre atividade desta ATPase e sensibilidade à crises agudas. Ademais, a administração de antagonistas EP1 e EP3 aboliu as alterações da atividade da Na+,K+-ATPase induzidas tanto por PTZ como por KA, de tal forma que estas parecem estar mais associadas com o fenômeno ictal em si, do que com os mecanismos de indução da crise. Contudo, os resultados mostram de forma clara que os receptores EP1 e EP3 podem se constituir possíveis novos alvos para o desenvolvimento de drogas antiepilépticas, pois antagonistas EP1 e EP3 diminuíram as crises, independente do agente convulsivante utilizado.

As mulheres na fase reprodutiva apresentam menor incidência de doenças cardiovasculares (DCV), em relação aos homens de mesma idade. Porém, essa vantagem desaparece na pós-menopausa, sugerindo que os hormônios sexuais femininos exercem algum efeito cardioprotetor. Um dos mecanismos propostos para explicar essa proteção é o fato dos estrógenos promoverem a produção de importantes fatores vasoativos pelo endotélio vascular, entre eles o óxido nítrico e a prostaglandina I2. Com a diminuição da quantidade de estrógenos circulante, as mulheres na pós-menopausa, estão mais suscetíveis à disfunção endotelial e a doenças cardiovasculares. Estudos têm demonstrado que a terapia de reposição hormonal (TRH) utilizada por mulheres na pós-menopausa combate os sintomas deste período, melhora o quadro de disfunção endotelial e o perfil lipídico e aumenta a síntese de fatores vasoativos, que auxiliam na prevenção da DCV. Entretanto a TRH vem sendo questionada por grandes estudos como o WHI (Writing Group for the Women\'s Health Initiative Investigators) e o HERS (Heart and Estrogen/Progestin Replacement Study), que mostraram um risco aumentado de desenvolvimento de câncer de mama e endometrial em mulheres fazendo uso da TRH. Entre as terapias alternativas para combater os sintomas indesejáveis da menopausa e as implicações mórbidas que acompanham esse período, sem expor as pacientes aos efeitos colaterais da TRH, a literatura aponta os fitoestrógenos, principalmente os extraídos da soja (Glycine max). O objetivo geral deste estudo é avaliar a ação das isoflavonas da soja, que vem sendo utilizadas por mulheres na pós-menopausa, na produção de óxido nítrico, prostaglandina E2 e endotelina-1, por células endoteliais, utilizando um modelo \"in vitro\", células endoteliais da linhagem ECV304. / During their reproductive years, women have a lower incidence of coronary heart disease (CHD) compared to men of similar age. However, this advantage disappears in post-menopause, suggesting that female sex hormones exert some cardio protective effect. One of the mechanisms proposed to explain this protection is the fact that estrogens promote the production of important vasoative factors by vascular endothelium, including nitric oxide and prostaglandin I2. By decreasing circulating estrogen, women in post-menopause are more susceptible of endothelial dysfunction and cardiovascular diseases. Studies have shown that the hormone replacement therapy (HRT) used by post-menopausal women in combating the symptoms of this period, improve endothelial dysfunction and lipid profile and increases the synthesis of vasoative factors, which help in the prevention of CHD. Meanwhile the HRT has been questioned by two large trials, the WHI (Writing Group for the Women\'s Health Initiative Investigators) and HERS (Heart and Estrogen/Progestin Replacement Study), which showed an increased risk of developing breast cancer and endometrial cancer in women using HRT. Among the alternative therapies to combat the symptoms of menopause and undesirable morbid implications that accompany this period, without exposing the patients to the side effects of HRT, the literature suggests the phytoestrogens, especially those from the soybean (Glycine max). The aim of this study is to evaluate the effect of isoflavones from soy, which are used by women in post-menopause, in the production of nitric oxide, prostaglandin E2 and endothelin-1 by endothelial cells, using an \"in vitro\" model: human endothelial cell line ECV304.

Introdução: Para realizar-se transplante de órgãos, é necessário que se preserve o enxerto durante a fase de isquemia fria. Entretanto, as soluções de preservação atuais não apresentam capacidade de oxigenar o tecido, o que causa, inevitavelmente, lesão celular. Em vista disso, busca-se associar substâncias antiinflamatórias, vasodilatadoras e antioxidantes à solução de preservação, tentando, assim, melhorar a qualidade da preservação hepática durante a fase de isquemia fria. Animais e métodos: Realizou-se a hepatectomia do doador em 36 ratos Wistar, divididos em 3 grupos de 12 animais. Os fígados desses ratos foram perfundidos e preservados durante 36 horas. No grupo 1, considerado grupo controle, foi utilizada a Solução da Universidade de Wisconsin (UW); o grupo 2 teve a UW acrescida de prostaglandina E1; o grupo 3 teve a UW acrescida de N-acetilcisteína. Realizaram-se biópsias hepáticas e coletaramse amostras da solução de preservação nos tempos de 12, 24 e 36 horas. Resultados: O estudo bioquímico da solução de preservação demonstrou que os níveis de transaminases se elevam com o passar do tempo, mas isso ocorreu em menor nível quando a UW foi acrescida de N-acetilcisteína. A análise histopatológica das lâminas das biópsias revelaram um infiltrado inflamatório portal menor quando a UW foi acrescida de prostaglandina E1. Conclusão: Assim, entendemos que a prostaglandina E1, considerada um potente antiinflamatório e vasodilatador, e a N-acetilcisteína, tida como um excelente antioxidante, acarretam efeito protetor ao enxerto hepático quando associadas à solução de preservação. / Introduction: In order to transplant an organ, it is necessary to preserve the graft during the cold ischemia period. The current preservation solutions are not able to oxygenate the tissue, which inevitably causes cell damage. We intend to associate non-inflammatory, vessel dilating and non-oxidative substances to the preservation solution to try to improve the quality of the liver preservation during the cold ischemia.Animals and methods: We performed the donor’s hepatectomy in 36 Wistar mice divided into 3 groups of 12 mice. The animals’ livers were perfused and preserved for 36 hours. In group 1, the control group, University of Wisconsin solution (UW) was used. In group 2, the UW was used together with prostaglandin 1 (0.5mcg/ml) and in group 3 UW was used together with N-acetylcysteine (0.3mg/ml). Hepatic biopsies were carried out, and samples of the preservation solution were collected in the preservation periods of 12, 24 and 36 hours. Results: The biochemical study of the preservation solution showed that the levels of transaminases increase over time, but this occurred less when UW was added to N-acetylcysteine (p>0,05). The hystopathological analysis of the biopsies showed a smaller portal inflammatory injury when UW was added to prostaglandin E1 (p<0,05). Conclusion: We therefore understand that prostaglandin E1, which is considered to be powerful non-inflammatory and vessel dilator, and Nacetylcysteine, which is an excellent non-oxidative substance, have a protective effect on the liver graft when associated with the preservation solution.

O presente estudo teve como objetivo avaliar a aplicação de análogo sintético da prostaglandina F2 (PGF2 ) associado à carbetocina ou ocitocina sobre a eficiência na indução ao parto em suínos. Foram analisados o tempo entre aplicação e início do trabalho de parto, duração do parto e percentual de natimortalidade. A indução do parto foi realizada aos 113 dias de gestação através da aplicação do análogo da PGF2 (cloprostenol sódico) via submucosa vulvar (SMV). As ocitocinas foram aplicadas 24 horas após a indução, pela via intramuscular (IM). O experimento 1 contou com 284 fêmeas em 4 tratamentos: T1- cloprostenol sódico; T2- cloprostenol sódico e 0,10 mg de carbetocina; T3- cloprostenol sódico e 10 UI de ocitocina; T4- solução salina 0,9% (NaCl) via SMV. O experimento 2 contou com 276 fêmeas em 4 tratamentos: T1- cloprostenol sódico; T2- cloprostenol sódico e 0,10 mg de carbetocina; T3- cloprostenol sódico e 0,05 mg de carbetocina; T4- cloprostenol sódico e 10 UI de ocitocina. Não houve diferença entre os tratamentos no número de leitões nascidos totais, nascidos vivos e percentual de partos com intervenção obstétrica manual. A sincronização dos partos é maior quando induzidos com cloprostenol comparado ao grupo não induzido. A utilização de cloprostenol associado à carbetocina resulta em menor duração do parto. / The present study aimed the analysis of a synthetic analogue of prostaglandin F2 (PGF2 ) associated to carbetocyn or oxytocin on the efficiency of farrowing induction in swine. The following variables were assessed: time between injections and start of farrowing, farrowing length and stillbirth percentage. Farrowing induction was performed at 113 days of gestation using injection of PGF2 analogue (sodium cloprostenol) by vulvar sub mucosal route (SMV). The oxytocins were used 24 hours after induction, by intra-muscular route (IM). Experiment 1 used 284 females in 4 treatments: T1-sodium cloprostenol; T2- sodium cloprostenol and 0.10 mg of carbetocyn; T3- sodium cloprostenol and 10 UI of oxytocin; T4- saline solution by SMV route. Experiment 2 used 276 females in 4 treatments: T1- sodium cloprostenol; T2- sodium cloprostenol and 0.10 mg of carbetocyn; T3- sodium cloprostenol and 0.05 mg of carbetocyn; T4- sodium cloprostenol and 10 UI of oxytocin. There was no difference between treatments regarding number of total born piglets per farrowing, born alive and percentage of farrowing using manual obstetrical intervention. Farrowing synchronization was higher when induced with cloprostenol when compared to the noninduced group. The use of cloprostenol associated with carbetocyn resulted in a diminished farrowing length.

Introdução: Para realizar-se transplante de órgãos, é necessário que se preserve o enxerto durante a fase de isquemia fria. Entretanto, as soluções de preservação atuais não apresentam capacidade de oxigenar o tecido, o que causa, inevitavelmente, lesão celular. Em vista disso, busca-se associar substâncias antiinflamatórias, vasodilatadoras e antioxidantes à solução de preservação, tentando, assim, melhorar a qualidade da preservação hepática durante a fase de isquemia fria. Animais e métodos: Realizou-se a hepatectomia do doador em 36 ratos Wistar, divididos em 3 grupos de 12 animais. Os fígados desses ratos foram perfundidos e preservados durante 36 horas. No grupo 1, considerado grupo controle, foi utilizada a Solução da Universidade de Wisconsin (UW); o grupo 2 teve a UW acrescida de prostaglandina E1; o grupo 3 teve a UW acrescida de N-acetilcisteína. Realizaram-se biópsias hepáticas e coletaramse amostras da solução de preservação nos tempos de 12, 24 e 36 horas. Resultados: O estudo bioquímico da solução de preservação demonstrou que os níveis de transaminases se elevam com o passar do tempo, mas isso ocorreu em menor nível quando a UW foi acrescida de N-acetilcisteína. A análise histopatológica das lâminas das biópsias revelaram um infiltrado inflamatório portal menor quando a UW foi acrescida de prostaglandina E1. Conclusão: Assim, entendemos que a prostaglandina E1, considerada um potente antiinflamatório e vasodilatador, e a N-acetilcisteína, tida como um excelente antioxidante, acarretam efeito protetor ao enxerto hepático quando associadas à solução de preservação. / Introduction: In order to transplant an organ, it is necessary to preserve the graft during the cold ischemia period. The current preservation solutions are not able to oxygenate the tissue, which inevitably causes cell damage. We intend to associate non-inflammatory, vessel dilating and non-oxidative substances to the preservation solution to try to improve the quality of the liver preservation during the cold ischemia.Animals and methods: We performed the donor’s hepatectomy in 36 Wistar mice divided into 3 groups of 12 mice. The animals’ livers were perfused and preserved for 36 hours. In group 1, the control group, University of Wisconsin solution (UW) was used. In group 2, the UW was used together with prostaglandin 1 (0.5mcg/ml) and in group 3 UW was used together with N-acetylcysteine (0.3mg/ml). Hepatic biopsies were carried out, and samples of the preservation solution were collected in the preservation periods of 12, 24 and 36 hours. Results: The biochemical study of the preservation solution showed that the levels of transaminases increase over time, but this occurred less when UW was added to N-acetylcysteine (p>0,05). The hystopathological analysis of the biopsies showed a smaller portal inflammatory injury when UW was added to prostaglandin E1 (p<0,05). Conclusion: We therefore understand that prostaglandin E1, which is considered to be powerful non-inflammatory and vessel dilator, and Nacetylcysteine, which is an excellent non-oxidative substance, have a protective effect on the liver graft when associated with the preservation solution.