Mechanisms of protease-activated receptor 2-evoked hyperexcitability of nociceptive neurons innervating the mouse colon

Agonists of protease-activated receptor 2 (PAR(2)) evoke hyperexcitability of dorsal root ganglia (DRG) neurons by unknown mechanisms. We examined the cellular mechanisms underlying PAR(2)-evoked hyperexcitability of mouse colonic DRG neurons to determine their potential role in pain syndromes such as visceral hyperalgesia. Colonic DRG neurons were identified by injecting Fast Blue and DiI retrograde tracers into the mouse colon. Using immunofluorescence, we found that DiI-labelled neurons contained PAR(2) immunoreactivity, confirming the presence of receptors on colonic neurons. Whole-cell current-clamp recordings of acutely dissociated neurons demonstrated that PAR(2) activation with a brief application (3 min) of PAR(2) agonists, SLIGRL-NH(2) and trypsin, evoked sustained depolarizations (up to 60 min) which were associated with increased input resistance and a marked reduction in rheobase (50% at 30 min). In voltage clamp, SLIGRL-NH(2) markedly suppressed delayed rectifier I(K) currents (55% at 10 min), but had no effect on the transient I(A) current or TTX-resistant Na(+) currents. In whole-cell current-clamp recordings, the sustained excitability evoked by PAR(2) activation was blocked by the PKC inhibitor, calphostin, and the ERK(1/2) inhibitor PD98059. Studies of ERK(1/2) phosphorylation using confocal microscopy demonstrated that SLIGRL-NH(2) increased levels of immunoreactive pERK(1/2) in DRG neurons, particularly in proximity to the plasma membrane. Thus, activation of PAR(2) receptors on colonic nociceptive neurons causes sustained hyperexcitability that is related, at least in part, to suppression of delayed rectifier I(K) currents. Both PKC and ERK(1/2) mediate the PAR(2)-induced hyperexcitability. These studies describe a novel mechanism of sensitization of colonic nociceptive neurons that may be implicated in conditions of visceral hyperalgesia such as irritable bowel syndrome.

Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways

The protease activated receptor-2 (PAR-2) belongs to a family of G-protein-coupled receptors that are activated by proteolysis. Trypsin cleaves PAR-2, exposing an N-terminal tethered ligand (SLIGRL) that activates the receptor. Messenger RNA (mRNA) for PAR-2 was found in guinea pig airway tissue by reverse transcription-polymerase chain reaction, and PAR-2 was found by immunohistochemistry in airway epithelial and smooth-muscle cells. In anesthetized guinea pigs, trypsin and SLIGRL-NH(2) (given intratracheally or intravenously) caused a bronchoconstriction that was inhibited by the combination of tachykinin-NK(1) and -NK(2) receptor antagonists and was potentiated by inhibition of nitric oxide synthase (NOS). Trypsin and SLIGRL-NH(2) relaxed isolated trachea and main bronchi, and contracted intrapulmonary bronchi. Relaxation of main bronchi was abolished or reversed to contraction by removal of epithelium, administration of indomethacin, and NOS inhibition. PAR-1, PAR-3, and PAR-4 were not involved in the bronchomotor action of either trypsin or SLIGRL-NH(2), because ligands of these receptors were inactive either in vitro or in vivo, and because thrombin (a PAR-1 and PAR-3 agonist) did not show cross-desensitization with PAR-2 agonists in vivo. Thus, we have localized PAR-2 to the guinea-pig airways, and have shown that activation of PAR-2 causes multiple motor effects in these airways, including in vivo bronchoconstriction, which is in part mediated by a neural mechanism.

Proteinase-activated receptor-2 (PAR2) agonist causes periodontitis in rats

Proteinase-activated receptor-2 (PAR2) is a G-protein-coupled receptor that mediates cellular responses to extracellular proteinases. Since PAR2 is expressed by oral epithelial cells, osteoblasts, and gingival fibroblasts, where its activation releases interleukin-8, we hypothesized that PAR2 activation may participate in periodontal disease in vivo. We investigated the role of PAR2 activation in periodontal disease in rats. Radiographic and enzymatic (myeloperoxidase) analysis revealed that topical application of PAR2 agonist causes periodontitis but also exacerbates existing periodontitis, leading to significant alveolar bone loss and gingival granulocyte infiltration. Inhibition of matrix metalloproteinase (MMP) and cyclo-oxygenase (COX) decreased PAR2 agonist-induced periodontitis. More specifically, the overexpression of COX-1, COX-2, MMP-2, and MMP-9 in gingival tissues suggests that they are involved in PAR 2-induced periodontitis. In conclusion, PAR2 agonist causes periodontitis in rats through a mechanism involving prostaglandin release and MMP activation. Inhibition of PAR2 may represent a novel approach to modulate host response in periodontitis.

Estudo geofísico regional sobre águas subterrâneas na Ilha do Marajó-Pará-Brasil

Nos últimos dez anos foram realizadas na parte leste da Ilha de Marajó (região dos campos naturais) pelo IDESP e NCGG, mais de 800 SEVs para fins hidrogeológicos. Na época, grande parte dessas SEVs não foram totalmente interpretadas em forma quantitativa, devido à falta de recursos técnicos para fazê-lo de forma eficiente. Agora, usando meios mais modernos para interpretação automática de SEVs, voltou-se a interpretá-las com a finalidade de apresentar uma visão regional dos principais aquíferos da área, agrupar as SEVs em famílias características, testar até que ponto essa interpretação é confiável e propor o modelamento bidimensional como técnica alternativa para interpretar as SEVs realizadas em certos locais da área em questão. Como resultado dessa interpretação, com base na teoria convencional dos meios estratificados, foram definidos três tipos de sistemas de aquíferos. 1. O primeiro, denominado de aquífero profundo, situado a profundidades maiores que 50m, estende-se por toda a região prospectada, estando provavelmente associada às camadas superiores da Formação Marajó ou às litologias altamente resistivas das camadas mais profundas do Grupo Pará. 2. O segundo, denominado de aquífero raso e de média profundidade, localiza-se na parte sul e sudeste da região a profundidades compreendidas entre 10 a 50m, e está associado às lentes arenosas do Grupo Pará. 3. O terceiro, é constituído pelos paleocanais e estruturas similares, distribuídos aleatoriamente na região a pouca profundidade. A partir do estudo detalhado das SEVs, decidiu-se classificá-las em 3 famílias características com seus respectivos tipos e apresentar mapas de localização e da espessura dos aquíferos, bem como mapas de condutância longitudinal total e resistividade média da área. Estes últimos, permitem que se divida a região dos campos da Ilha de Marajó em três zonas principais: 1. Uma, altamente resistiva, situada ao sul e sudeste, a qual coincide com os terrenos aflorantes do Grupo Pará. 2. Outra, altamente condutiva, está localizada no centro e norte, onde se encontram aleatoriamente distribuídos os paleocanais e coincide com os terrenos topograficamente mais baixos, geralmente argilosos e embebidos de água salgada, que são procedentes da erosão dos terrenos circundantes topograficamente mais altos. 3. A última é medianamente resistiva e está relacionada com os terrenos vizinhos à cidade de Chaves (noroeste da região dos campos), os quais apresentam semelhanças com os do sul e sudeste da área. Usando-se a técnica de inversão na interpretação de uma SEV característica de cada família, testou-se, através do seu tratamento estatístico, até que ponto os modelos usados na interpretação dessas SEVs (teoria convencional dos meios estratificados) seriam confiáveis. Conclui-se, então, que a alta correlação existente entre os parâmetros dos modelos assumidos (camadas horizontais, isotrópicas e homogêneas) pode-se dever à utilização de modelos geofísicos muito simples para interpretar a complexa geologia de Marajó. Tendo-se verificado que nem sempre é possível aplicar a teoria das SEVs em meios horizontalmente estratificados para interpretar SEVs obtidas em certos locais de Marajó, os quais muitas vezes apresentam bruscas variações laterais de resistividade, passou-se a demonstrar que estas variações laterais afetam profundamente os dados das SEVs, utilizando-se para isto a técnica dos elementos finitos, a qual leva em conta essa variação bidimensional das propriedades físicas do meio. Foi também possível com esta técnica, modelar uma estrutura rasa, semelhante a um paleocanal, concluindo-se que estes resultados sugerem o emprego, duma forma mais profunda, deste tipo de tratamento para os dados obtidos na região dos campos da Ilha de Marajó.

Down-regulation of protease activated receptor 2, interleukin-17 and other pro-inflammatory genes by subantimicrobial doxycycline dose in a rat periodontitis model

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Human beta-defensin 2 and protease activated receptor-2 expression in patients with chronic periodontitis

Objective: Some previous studies have shown that gingipains, trypsin-like proteases produced by Porphyromonas gingivalis, up-regulate human beta defensin-2 (HBD-2) mRNA expression through protease-activated receptor-2 (PAR(2)) in gingival epithelial cells. This study aimed at investigating salivary HBD-2 levels and crevicular PAR(2) mRNA expression in human chronic periodontitis and evaluating whether periodontal treatment affected this process. Methods: Salivary and gingival crevicular fluid (GCF) samples were collected from periodontally healthy (control) and chronic periodontitis patients at baseline and 50 days after nonsurgical periodontal treatment. Salivary HBD-2, and GCF TNF-alpha levels were analysed by ELISA, and PAR(2) mRNA at the GCF was evaluated by RT-PCR. Results: P. gingivalis was significantly (p < 0.05) more prevalent in patients with chronic periodontitis when compared to controls. This prevalence decreased after periodontal therapy (p < 0.0001). The control group showed statistically significant lower levels of HBD-2, TNF-alpha, and PAR(2) expression when compared to the chronic periodontitis group. In addition, periodontal treatment significantly reduced PAR(2) expression and HBD-2 levels in chronic periodontitis patients (p < 0.001). Conclusions: Our results suggest that salivary HBD-2 levels and PAR(2) mRNA expression from GCF are higher in subjects with chronic periodontitis than in healthy subjects, and that periodontal treatment decreases both HBD-2 levels and PAR(2) expression. (C) 2012 Elsevier Ltd. All rights reserved.

Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E2 and F1α from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.

Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells

The proteinase-activated receptor 2 (PAR-2) expression is increased in endothelial cells derived from women with preeclampsia, characterized by widespread maternal endothelial damage, which occurs as a consequence of elevated soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; commonly known as sFlt-1) in the maternal circulation. Because PAR-2 is upregulated by proinflammatory cytokines and activated by blood coagulation serine proteinases, we investigated whether activation of PAR-2 contributed to sVEGFR-1 release. PAR-2–activating peptides (SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2) and factor Xa increased the expression and release of sVEGFR-1 from human umbilical vein endothelial cells. Enzyme-specific, dominant-negative mutants and small interfering RNA were used to demonstrate that PAR-2–mediated sVEGFR-1 release depended on protein kinase C-ß1 and protein kinase C-e, which required intracellular transactivation of epidermal growth factor receptor 1, leading to mitogen-activated protein kinase activation. Overexpression of heme oxygenase 1 and its gaseous product, carbon monoxide, decreased PAR-2–stimulated sVEGFR-1 release from human umbilical vein endothelial cells. Simvastatin, which upregulates heme oxygenase 1, also suppressed PAR-2–mediated sVEGFR-1 release. These results show that endothelial PAR-2 activation leading to increased sVEGFR-1 release may contribute to the maternal vascular dysfunction observed in preeclampsia and highlights the PAR-2 pathway as a potential therapeutic target for the treatment of preeclampsia.

Aflatoxinas e ocratoxina A em alimentos e riscos para a saúde humana

OBJETIVOS: A presença de micotoxinas em alimentos tem sido correlacionada a várias patologias humanas, e as autoridades de saúde no mundo todo têm implementado ações para diminuir a ingestão desses compostos pela dieta. Realizou-se pesquisa para analisar os níveis de aflatoxinas e ocratoxina A de alimentos para consumo e avaliar o potencial de risco da exposição humana a essas micotoxinas. MÉTODOS: Foram analisadas 366 amostras de alimentos consumidos no Distrito Federal, no período de julho de 1998 a dezembro de 2001, como amendoim e derivados, castanhas, milho, produtos de trigo e/ou aveia, arroz e feijão. As amostras foram processadas, e as micotoxinas extraídas, detectadas e quantificadas por fluorescência após separação em cromatografia camada delgada. RESULTADOS: Foram detectadas aflatoxinas em 19,6% das amostras, em amendoim cru e derivados, milho de pipoca, milho em grão e castanha-do-pará (>2 mig/kg). Amendoim e derivados apresentaram maior incidência de contaminação por aflatoxinas (34,7%) com amostras contendo até 1.280 mig/kg de AFB1+AFG1 e 1.706 mig/kg de aflatoxinas totais. Das amostras positivas, AFB1 estava presente em 98,5%, AFB2 em 93%, AFG1 em 66,7% e AFG2 em 65,4%. A ocratoxina A não foi detectada (<25 mig/kg) em nenhuma amostra analisada. CONCLUSÃO: Os níveis de contaminação encontrados em amendoim e derivados ultrapassaram os níveis máximos permitidos pela legislação brasileira, podendo significar fator de risco para a população que os consome regularmente. A conscientização dos produtores de alimentos e as ações de vigilância sanitária permanentes são essenciais para diminuir a exposição humana a esses compostos e prevenir doenças crônicas advindas dessa exposição.

Melanosome transfer between melanocytes and keratinocytes

RESUMO: A pele é o maior órgão do corpo humano e a sua pigmentação é essencial para a sua coloração e proteção contra os efeitos nocivos da radiação ultravioleta (UV). A pigmentação da pele resulta essencialmente de três processos: a síntese e o armazenamento de melanina pelos melanócitos, em organelos especializados denominados melanossomas; o transporte dos melanossomas dentro dos melanócitos; e finalmente, a transferência dos melanossomas para os queratinócitos adjacentes. Nos queratinócitos, a melanina migra para a região perinuclear apical da célula para formar um escudo protetor,responsável pela proteção do DNA dos danos causados pela radiação UV. Os melanócitos estão localizados na camada basal da epiderme e contactam com 30-40 queratinócitos. Em conjunto, estas células formam a “unidade melano-epidérmica”. Apesar dos processos de síntese e transporte de melanina nos melanócitos estarem bastante bem caracterizados, os mecanismos moleculares subjacentes à transferência inter-celular de melanina são menos conhecidos e ainda controversos. Dados preliminares obtidos pelo nosso grupo, que se basearam na observação de amostras de pele humana por microscopia electrónica, indicam que a forma predominante de transferência de melanina na epiderme consiste na exocitose dos melanossomas pelos melanócitos e subsequente endocitose da melanina por queratinócitos. Para além disso sabe-se que as proteínas Rab, que controlam o tráfego membranar, estão envolvidas em várias etapas de pigmentação da pele, nomeadamente na biogénese e no transporte de melanina. Assim, dado o seu papel fundamental nestes processos, questionámo-nos sobre o seu envolvimento na transferência de melanina. Com este trabalho, propomo-nos a expandir o conhecimento atual sobre a transferência de melanina na pele, através do estudo detalhado dos seus mecanismos moleculares, identificando as proteínas Rab que regulam o processo. Pretendemos também confirmar o modelo de exo/endocitose como sendo o mecanismo principal de transferência de melanina. Primeiro, explorámos a regulação da secreção de melanina pelos melanócitos e analisámos o papel de proteínas Rab neste processo. Os resultados foram obtidos recorrendo a um método in vitro, desenvolvido previamente no laboratório, que avalia a quantidade de melanina segregada para o meio de cultura por espectrofotometria, e ainda por microscopia, contando o número de melanossomas transferidos para os queratinócitos. Através de co-culturas de melanócitos e queratinócitos, verificou-se que os queratinócitos estimulam a libertação de melanina dos melanócitos para o meio extra-celular, bem como a sua transferência para os queratinócitos. Além disso, a proteína Rab11b foi identificada como um regulador da exocitose de melanina e da sua transferência para os queratinócitos. De facto, a diminuição da expressão de Rab11b em melanócitos provocou a redução da secreção de melanina estimulada por queratinócitos, bem como da transferência desta. Em segundo lugar, para complementar o nosso estudo, centrámos a nossa investigação na internalização de melanina por queratinócitos. Especificamente, usando uma biblioteca de siRNA, explorámos o envolvimento de proteínas Rab na captação de melanina por queratinócitos. Como primeira abordagem, usámos esferas fluorescentes como substituto de melanina, avaliando os resultados por citometria de fluxo. No entanto, este método revelou-se ineficaz uma vez que a internalização destas esferas é independente do recetor PAR-2 (recetor 2 ativado por protease), que foi previamente descrito como essencial na captação de melanina por queratinócitos Posteriormente, foi desenvolvido um novo protocolo de endocitose baseado em microscopia, usando melanossomas sem a membrana envolvente (melanocores) purificados do meio de cultura de melanócitos, incluindo um programa informático especialmente desenhado para realizar uma análise semi-automatizada. Após internalização, os melanocores acumulam-se na região perinuclear dos queratinócitos, em estruturas que se assemelham ao escudo supranuclear observado na pele humana. Seguidamente, o envolvimento do recetor PAR-2 na captação de melanocores por queratinócitos foi confirmado, utilizando o novo protocolo de endocitose desenvolvido. Para além disso, a necessidade de quatro proteínas Rab foi identificada na internalização de melanocores por queratinócitos. A redução da expressão de Rab1a ou Rab5b em queratinócitos diminuiu significativamente o nível de internalização de melanocores, enquanto o silenciamento da expressão de Rab2a ou Rab14 aumentou a quantidade de melanocores internalizados por estas células. Em conclusão, os resultados apresentados corroboram as observações anteriores, obtidas em amostras de pele humana, e sugerem que o mecanismo de transferência predominante é a exocitose de melanina pelos melanócitos, induzida por queratinócitos, seguida por endocitose pelos queratinócitos. A pigmentação da pele tem implicações tanto ao nível da cosmética, como ao nível médico, relacionadas com foto-envelhecimento e com doenças pigmentares. Assim sendo, ao esclarecer quais os mecanismos moleculares que regulam a transferência de melanina na pele, este trabalho pode conduzir ao desenvolvimento de novas estratégias para modular a pigmentação da pele.----------------ABSTRACT: Skin pigmentation is achieved through the highly regulated production of the pigment melanin in specialized organelles, termed melanosomes within melanocytes. These are transported from their site of synthesis to the melanocyte periphery before being transferred to keratinocytes where melanin forms a supra-nuclear cap to protect the DNA from UVinduced damage. Together, melanocytes and keratinocytes form a functional complex, termed “epidermal-melanin unit”, that confers color and photoprotective properties to the skin. Skin pigmentation requires three processes: the biogenesis of melanin; its intracelular transport within the melanocyte to the cell periphery; and the melanin transfer to keratinocytes. The first two processes have been extensively characterized. However, despite significant advances that have been made over the past few years, the mechanisms underlying inter-cellular transfer of pigment from melanocytes to keratinocytes remain controversial.Preliminary studies from our group using electron microscopy and human skin samples found evidence for a mechanism of coupled exocytosis-endocytosis. Rab GTPases are master regulators of intracellular trafficking and have already been implicated in several steps of skin pigmentation. Thus, we proposed to explore and characterize the molecular mechanisms of melanin transfer and the role of Rab GTPases in this process. Moreover, we investigated whether the exo/endocytosis model is the main mechanism of melanin transfer. We first focused on melanin exocytosis by melanocytes. Then, we started to investigate the key regulatory Rab proteins involved in this step by establishing an in vitro tissue culture model of melanin secretion. Using co-cultures of melanocytes and keratinocytes, we found that keratinocytes stimulate melanin release and transfer. Moreover, depletion of Rab11b decreases keratinocyte-induced melanin exocytosis by melanocytes. In order to determine whether melanin exocytosis is a predominant mechanism of melanin transfer, the amount of melanin transferred to keratinocytes was then assayed in conditions where melanin exocytosis was inhibited. Indeed, Rab11b depletion resulted in a significant decrease in melanin uptake by keratinocytes. Taken together, these observations suggest that Rab11b mediates melanosome exocytosis from melanocytes and transfer to keratinocytes. To complement and extend our study, we of melanin by keratinocytes. Thus, we aimed to explore the effect of depleting Rab GTPases on melanin uptake and trafficking within keratinocytes. As a first approach, we used fluorescent microspheres as a melanin surrogate. However, the uptake of microspheres was observed to be independent of PAR-2, a receptor that is required for melanin uptakecentred our attention in the internalization of melanin by keratinocytes. Thus, we aimed to explore the effect of depleting Rab GTPases on melanin uptake and trafficking within keratinocytes. As a first approach, we used fluorescent microspheres as a melanin surrogate. However, the uptake of microspheres was observed to be independent of PAR-2, a receptor that is required for melanin uptake.Therefore, we concluded that microspheres were uptaken by keratinocytes through a different pathway than melanin. Subsequently, we developed a microscopy-based endocytosis assay using purified melanocores (melanosomes lacking the limiting membrane) from melanocytes, including a program to perform a semi-automated analysis. Melanocores are taken up by keratinocytes and accumulate in structures in the perinuclear area that resemble the physiological supranuclear cap observed in human skin. We then confirmed the involvement of PAR-2 receptor in the uptake of melanocores by keratinocytes, using the newly developed assay. Furthermore, we identified the role of four Rab GTPases on the uptake of melanocores by keratinocytes. Depletion of Rab1a and Rab5b from keratinocytes significantly reduced the uptake of melanocores, whereas Rab2a, and Rab14 silencing increased the amount the melanocores internalized by XB2 keratinocytes. In conclusion, we present evidence supporting keratinocyte-inducedmelanosome exocytosis from melanocytes, followed by endocytosis of the melanin core by keratinocytes as the predominant mechanism of melanin transfer in skin. Although advances have been made, there is a need for more effective and safer therapies directed at pigmentation disorders and also treatments for cosmetic applications. Hence, the understanding of the above mechanisms of skin pigmentation will lead to a greater appreciation of the molecular machinery underlying human skin pigmentation and could interest the pharmaceutical and cosmetic industries.

50 anos de pesquisas em limnologia na Amazônia

Constitui-se o presente trabalho da transcrição integral da palestra realizada pelo Dr. Harald Sioli (1910-2004) no Seminário da Amazônia, em 10 de agosto de 1990, quando numa última viagem ao Amazonas, ao completar 80 anos de idade, foi homenageado pelos colegas e a convite do INPA falou sobre "50 anos de pesquisas em limnologia na Amazônia" que, segundo suas próprias palavras, foram "os dias mais decisivos de minha vida". O texto pode ser dividido em duas partes: a primeira se refere à história pessoal - da chegada ao Brasil, de como descobriu a Amazônia e sobreviveu, no interior do Amazonas e Pará, à 2.ª Guerra Mundial, até se tornar pesquisador do INPA e firmar o Convênio INPA/Max Planck. A segunda parte fala da sua pesquisa em limnologia, fazendo contrapontos com pesquisa e pesquisadores brasileiros e estrangeiros, os do passado e os contemporâneos, assim como da convivência com os caboclos que, no seu entender, "vivem com a floresta e não contra a floresta". A transcrição da palestra, foi feita por Elci Silva, e Antonio Alvarez e preservada por Terezinha Soares, então lotados na Assessoria de Tecnologia e Extensão - ASTE que à época organizava o Seminário da Amazônia. A publicação foi autorizada pessoalmente pelo Dr. Sioli, ainda em 2000, através de correspondência em que ele afirmava "ficarei muito satisfeito se ainda puder ser um pouco útil na luta pela sobrevivência da Amazônia, daquela minha segunda pátria". Está, enfim, sendo publicada. E ainda que Haraldi Sioli já não esteja entre nós, representa uma homenagem da Acta Amazonica à memória daquele que dedicou tantos anos ao conhecimento e à preservação da Amazônia.

Expression of protease-activated receptors in arthritic synovial tissues

Clinical and experimental evidence suggests that synovial thrombin formation in arthritic joints is prominent and deleterious, leading to exacerbation of rheumatoid arthritis (RA). In this context, cellular effects of thrombin mediated by the protease-activated receptors (PARs) in arthritic joints may be of paramount significance. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin whereas PAR2 is activated by trypsin and few other proteases.We first explored PARs expression in RA synovial tissues. Synovial membranes from 11 RA patients were analyzed for PARs expression by RT-PCR and by immunohistology. PAR4 was found in all the biopsies, whereas the expression of PAR1, PAR 2 and PAR3 was more restricted (8/11, 5/11 and 3/11 respectively). In the arthritic synovial membrane of murine antigen-induced arthritis (AIA) we found coexpression of the four different PARs. Next, we explored the functional importance of PAR1 during AIA in vivo using PAR-1 deficient mice. The phenotype of PAR1-deficient mice (n = 22), based on the analysis of arthritis severity (as measured by 99 m tecnetium uptake, histological scoring and intra-articular fibrin measurements) was similar to that of wild-type mice (n = 24). In addition, the in vivo production of antibodies against mBSA was also similar. By contrast, the mBSA-induced in vitro lymph node cell proliferation was significantly decreased in PAR1-deficient mice as compared with controls. Accordingly, mBSA-induced production of interferon-γ by lymph node cells in culture was significantly decreased in PAR1-deficient mice as compared with controls, whereas opposite results were observed for production of IL-10.

Surrogate markers for atheriosclerosis in overweight subjects with atherogenic dyslipidemia : the GEMS project

Rapport de synthèseLe syndrome métabolique (défini par les critères ATP III par la présence au minimum de 3 des facteurs suivants : taux plasmatiques d'HDL-cholestérol < 1,04 mmol/1 chez l'homme et < 1.29 mmol/1 chez la femme, taux plasmatiques de triglycérides > 1,69 mmol/1, tension artérielle > 130/85 mmHg, glycémie >6,1 mmol/1, tour de taille > 108 cm chez l'homme et > 88 cm chez la femme) représente une constellation de facteurs de risque majeurs pour le développement de maladies cardiovascu-laires. Il n'est pas encore établi actuellement quelle composante de ce syndrome contribue de manière plus marquée au risque de développer une athérosclérose. Dans le but d'éclaircir la pathogenèse de ce syndrome, une étude multicentrique intitulée GEMS (« Genetic Epidemiology of Metabolic Syndrome ») a été initiée afin de déterminer si la constellation d'une dyslipidémie avec HDL-C bas et TG élevé est un marqueur sensible de l'homogénéité génétique chez les individus atteints de syndrome métabolique.Dans l'étude menée à Lausanne (multicentrique), la contribution de la dyslipidémie avec HDL-C bas et TG élevé dans la pathogenèse de l'athérosclérose a été évaluée par 2 examens, reconnus comme marqueurs fiables de la vasculopathie : la mesure de l'épaisseur intima média carotidienne par ultrasonographic et l'évaluation de la dysfonction endothéliale de la microcirculation cutanée. Deux groupes de sujets comparables en terme d'âge et de sexe et souffrant d'un excès pondéral (BMI > 25 kg/m2) mais normoglycémiques ont été comparés. Ces deux groupes (étude cas-témoins) étaient uniquement discordants quant à leurs profils lipidiques. Ainsi, 120 cas, définis comme ayant un HDL-cholestérol bas (< 25 percentile pour l'âge et le sexe dans la population générale) et des TG élevés (> 75 percentile) ont été comparés à 120 contrôles avec un HDL-cholestérol haut (> 50 percentile) et des TG bas (< 50 percentile). Un doppler des artères carotides et fémorales a été effectué pour déterminer l'épaisseur de l'intima média et la présence ou non de plaques d'athérome. La fonction endothéliale a été évaluée par un laser doppler sur la micro-circulation cutanée (réponse hyperémique à une occlusion transitoire de la circulation de l'avant-bras par une manchette à pression et mesure de la vasodilatation induite par un échauffement local de la peau avec de l'eau). Un enregistrement de la pression artérielle ambulatoire sur la journée (Remler) a été pratiqué chez tous les sujets.Les résultats obtenus montrent que les cas ont une prévalence plus élevée de plaques d'athérome (médiane 1,5 ± 0,15 vs 0,8 > 0,15, p<.001), une épaisseur intima média plus importante (médiane 0,66 ± 0,15 vs 0,61 ± 0,15, p<.01), ainsi qu'une réduction significative de la vasodilatation endothéliale induite par la chaleur et post-ischémique comparativement aux contrôles.En conclusion, le profil lipidique associant un HDL-cholestérol bas et des triglycérides élevés représente un risque majeur de développer une maladie athéromateuse périphérique et est associée à une augmentation de l'épaisseur intima média et une altération de la fonction endothéliale chez les individus en surcharge pondérale. Bien qu'un HDL-cholestérol bas soit fréquemment associé à une hypertriglycéridémie, les résultats de notre étude peuvent suggérer un rôle potentiel de la fraction HDL-cholestérol comme un puissant agent anti-athérogénique.

Epithelial effects of proteinase-activated receptors in the gastrointestinal tract

The intestinal epithelium plays a crucial role in providing a barrier between the external environment and the internal milieu of the body. A compromised mucosal barrier is characteristic of mucosal inflammation and is a key determinant of the development of intestinal diseases such as Crohn's disease and ulcerative colitis. The intestinal epithelium is regularly exposed to serine proteinases and this exposure is enhanced in numerous disease states. Thus, it is important to understand how proteinase-activated receptors (PARs), which are activated by serine proteinases, can affect intestinal epithelial function. This review surveys the data which demonstrate the wide distribution of PARs, particularly PAR-1 and PAR-2, in the gastrointestinal tract and accessory organs, focusing on the epithelium and those cells which communicate with the epithelium to affect its function. PARs have a role in regulating secretion by epithelia of the salivary glands, stomach, pancreas and intestine. In addition, PARs located on subepithelial nerves, fibroblasts and mast cells have important implications for epithelial function. Recent data outline the importance of the cellular site of PAR expression, as PARs expressed on epithelia may have effects that are countered by PARs expressed on other cell types. Finally, PARs and their ability to promote epithelial cell proliferation are discussed in terms of colon cancer.