Mechanisms of the anti-obesity effects of oxytocin in diet-induced obese rats.

Apart from its role during labor and lactation, oxytocin is involved in several other functions. Interestingly, oxytocin- and oxytocin receptor-deficient mice develop late-onset obesity with normal food intake, suggesting that the hormone might exert a series of beneficial metabolic effects. This was recently confirmed by data showing that central oxytocin infusion causes weight loss in diet-induced obese mice. The aim of the present study was to unravel the mechanisms underlying such beneficial effects of oxytocin. Chronic central oxytocin infusion was carried out in high fat diet-induced obese rats. Its impact on body weight, lipid metabolism and insulin sensitivity was determined. We observed a dose-dependent decrease in body weight gain, increased adipose tissue lipolysis and fatty acid β-oxidation, as well as reduced glucose intolerance and insulin resistance. The additional observation that plasma oxytocin levels increased upon central infusion suggested that the hormone might affect adipose tissue metabolism by direct action. This was demonstrated using in vitro, ex vivo, as well as in vivo experiments. With regard to its mechanism of action in adipose tissue, oxytocin increased the expression of stearoyl-coenzyme A desaturase 1, as well as the tissue content of the phospholipid precursor, N-oleoyl-phosphatidylethanolamine, the biosynthetic precursor of the oleic acid-derived PPAR-alpha activator, oleoylethanolamide. Because PPAR-alpha regulates fatty acid β-oxidation, we hypothesized that this transcription factor might mediate the oxytocin effects. This was substantiated by the observation that, in contrast to its effects in wild-type mice, oxytocin infusion failed to induce weight loss and fat oxidation in PPAR-alpha-deficient animals. Altogether, these results suggest that oxytocin administration could represent a promising therapeutic approach for the treatment of human obesity and type 2 diabetes.

Mécanisme moléculaire de protection des HDLs contre le stress du réticulum endoplasmique dans la cellule beta pancréatique. [Molecular mechanisms of HDLs to protect against the endoplasmic reticulum stress in pancreatic beta cell.]

Introduction: Les particules de HDL (High Density Lipoproteins) ont des fonctions très diverses notamment anti-inflamatoires, anti-apoptotiques ou anti-oxydatives. Chez les patients diabétiques, les niveaux de HDLs sont bas, les prédisposants ainsi à un risque élévé à développer une maladie cardiovasculaire. Sachant que le s HDLs ont également un effet protecteur sur la cellule beta, le but de cette étude est dinvestigué les mécanismes moléculaires de cette protection contre le stress du réticulum, stress qui contriubue au développement du diabéte de type 2. Résultats: La thapsigargine et la tunicamycine induisent lapoptose en induisant un stress dans le réticulum endoplasmique (RE) par un mauvais repliement des protéines dans le RE, ainsi que l'activation de l'UPR (Unfolded Protein Respons) avec trois voies communes de signalisation intracellulaire (IRE1, PREK et ATF6). Ces voix veillent tout d'abord à augmenter la capacité de repliement des protéines et le cas échéant à lapoptose. Nos résultats montrent que les HDLs sont capable d'inhuber lapoptose induite par la thapsigargine et la tunicamycine dans les MIN6. Dans le cas du traitement avec la thapsigargine, plusieurs marqueurs des voix UPR sont bloqués en présence des HDLs, suggérant que l'effet anti-apoptotiques des HDLs s'exerce au niveau ou en amont du RE. Les HDLS par contre ne bloquent par la sortie de calcium du RE induite par la thapsigargine ce qui indique que les HDLs n'interfèrent pas avec l'action de cette drogue sur sa cible (SERCA). Dans le cas de la la tunicamycine, les HDLs ne bloquent pas, ou très légèrement, l'activation des voix de l'UPR. La protection induite par les HDLs contre la mort engendrée par la tunicamycine s'sexerce dont apparement en aval de l'UPR et reste à être déterminer. Conclusions: Nos données suggérent que les HDLs sont capable de protéger la cellule beta contre le stress du réticulum mais apparement de façon différente selon les modalités d'inductions de ce stress.

Molecular mechanisms of insulin exocytosis role of small monomeric GTP-ASES

SUMMARYInsulin secretion from pancreatic beta-cells is a fundamental condition for the maintenance of blood glucose levels. During the last decades, important components of the molecular machinery controlling hormone release have been characterized. My PhD thesis was dedicated to the study of new signaling pathways regulating insulin exocytosis and in particular to the role of small monomelic guanine triphosphatase or GTPases controlling the last events of hormone release.The first part of my thesis focused on Ras-like (Ral) RalA and RalB proteins. We investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed their impact on different steps of the insulin-secretory process. Our results have shown that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-IE cells by RNA interference led to a decrease in secretagogue-induced hormone release. The activation of the GTPase, followed by FRET imaging, is triggered by increases in intracellular Ca and cAMP. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane, detected by TIRF microscopy and with strong impairment in PLD1 activation in response to secretagogues. RalA was found to be activated by the exchange factor RalGDS, which regulates hormone secretion induced by secretagogues and the docking step of insulin-containing granules at the plasma membrane. In the second part of this work we have shown that a member of the Rab family, Rab37, is present on insulin-containing secretory granules of pancreatic beta-cells. In addition, our experiments have suggested that Rab37 is required to obtain an optimal insulin secretory response induced by secretogogues and is important for the docking step of insulin-containing granules at the plasma membrane.

A new species of Scymnobius Casey (Coleoptera, Coccinellidae, Scymnini) from Pernambuco, Brazil

A new species of Scymnobius Casey (Coleoptera, Coccinellidae, Scymnini) from Pernambuco, Brazil. Scymnobius pernambucensis sp. nov. from Pernambuco, Brazil, is described and illustrated. This is the third species of this genus recorded from Brazil.

Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

Constitutively active G protein-coupled receptors: potential mechanisms of receptor activation.

Mutations of G protein-coupled receptors can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The analysis of the constitutively active G protein-coupled receptors has provided important informations about the molecular mechanisms underlying receptor activation and drug action.

Setting in motion the immune mechanisms underlying genetically determined resistance and susceptibility to infection with Leishmania major.

The murine model of infection with Leishmania major has allowed the demonstration of a causal relationship between, on the one hand, genetically determined resistance to infection and the development of a Th1 CD4+ cell response, and on the other hand, genetically determined susceptibility and Th2 cell maturation. Using this murine model of infection, the role of cytokines in directing the functional differentiation pathway of CD4+ T cell precursors, has been demonstrated in vivo. Thus, IL-12 and IFN-gamma have been shown to favour Th1 cell development and IL-4 is crucial for the differentiation of Th2 responses. Maturation of a Th2 response in susceptible BALB/c mice following infection with L. major is triggered by the IL-4 produced during the first two days after parasite inoculation. This IL-4 rapidly renders parasite specific CD4+ T cells precursors unresponsive to IL-12. A restricted population of CD4+ T cells expressing the V beta 4V alpha 8 TCR heterodimer and recognizing a single epitope on the LACK (Leishmania Activated C-Kinase) antigen of L. major is responsible for this rapid production of IL-4, instructing subsequent differentiation towards the Th2 phenotype of CD4+ T cells specific for several parasite antigens.

Caracterização de solos com a chernozêmico na Zona da Mata norte do estado de Pernambuco

O presente trabalho, realizado, em 1995, no município de Nazaré da Mata, teve como objetivo caracterizar morfológica, física, química, mineralógica e micromorfologicamente perfis com horizonte A chernozêmico na Zona da Mata Norte de Pernambuco. Foram descritos morfologicamente e coletados três perfis de solo com horizontes A chernozêmico, pertencentes às seguintes classes: Solo Litólico, Brunizém Avermelhado e Podzólico Vermelho-Amarelo, e realizadas análises físicas, químicas (incluindo quantificação das frações húmicas), mineralógicas e pedográficas. Os atributos pedológicos e as condições ambientais permitiram estabelecer a hipótese de que os horizontes superficiais têm sua formação ligada a interações de clima e vegetação, haja vista que, apesar das condições tropicais chuvosas, esses solos mantêm boa reserva química, provavelmente, graças à ocorrência de uma estação mais seca, que se estende por 5 meses, a qual provoca a queda das folhas e reduz a lixiviação de bases, conferindo, assim, significativa incorporação do material orgânico. Tal reserva química parece contribuir para a preservação da mica (biotita) nesses horizontes, a qual, ante as condições tropicais chuvosas, degradar-se-ia. Entretanto, a riqueza química da solução do solo refrearia o processo intempérico, na medida em que a existência do potássio na solução circundante às partículas sólidas minerais não ocasionaria a retirada do referido elemento da estrutura cristalina da biotita. Dessa feita, tal mineral se mantém preservado no ambiente pedológico. Qualitativamente, as diferenças observadas nas frações húmicas mostram-se decorrentes da influência da erosão, sendo encontrados maiores valores de frações menos resistentes no perfil mais erodido (Solo Litólico). Os horizontes subsuperficiais, por sua vez, são caracterizados, inicialmente, por um processo de formação de argila in situ, sendo compostos de alguma caulinita e interestratificados irregulares envolvendo minerais do tipo 2:1 e 1:1. Com a continuidade da pedogênese, tais horizontes são dominados por minerais mais estáveis (caulinitização), havendo movimentação da fração mais fina, o que leva à formação do horizonte B iluvial, onde ocorre também a formação de argila in situ Fatores como variação quantitativa da mineralogia do material de origem, padrão estrutural do gnaisse e relevo, parecem controlar a formação e evolução dos solos estudados.

Potencial de mineralização de N e de C em vinte solos de Pernambuco

A capacidade de mineralização de C e de N de 20 solos de Pernambuco (10 da zona úmida e 10 da semi-árida) foi determinada em casa de vegetação, entre janeiro e outubro de 1987, incubando-se amostras de 50 g, das profundidades de 0-20 e 20-40 cm, e medindo-se, periodicamente, durante 20 e 4 semanas, respectivamente, o C-CO2 liberado e o N mineral (NO3- e NH4+) acumulado. As quantidades mineralizadas variaram muito entre os solos, mas, apesar disto, foram maiores na zona úmida que na semi-árida e, na semi-árida, foram maiores na camada de 0-20 que na de 20-40 cm. Os solos apresentaram distintos padrões de mineralização ao longo da incubação, com a fase inicial com retardo ou com grande mineralização e a final com estabilização ou queda progressiva. As curvas de C tenderam mais à estabilização final que as de N. As quantidades de C e de N mineralizados foram bem relacionadas entre si e com os teores de C e N totais do solo e as de N mineralizado com as quantidades absorvidas por plantas cultivadas em potes com os mesmos solos. A matéria orgânica da zona úmida tendeu a ser mais lábil que a da semi-árida, principalmente quanto ao N, e a da camada superficial mais que a da subsuperficial.

The yield of high-density electric source imaging in focal epilepsy

Background: EEG is the cornerstone of epilepsy diagnostics and mandatory to determine the underlying epilepsy syndrome (e.g. focal vs idiopathic generalized). However, its potential as imaging tool is still underrecognized. In the present study, we aim to determine the prerequisites of maximal benefit of electric source imaging (ESI) to localize the irritative zone in patients with focal epilepsy. Methods: 150 patients suffering from focal epilepsy and with minimum 1 year post-operative follow-up were studied prospectively by reviewers blinded to the underlying diagnosis and outcome. We evaluated the influence of two important factors on sensitivity and specificity of ESI: the number of electrodes (low resolution, LR-ESI: \30 vs. high resolution, HR-ESI: 128-256 electrodes), and the use of individual MRI (i-MRI) vs. template MRI (t-MRI) as head model.Results: ESI had a sensitivity of 85% and a specificity of 87% when HR-ESI with i-MRI was used. Using LR-ESI, sensitivity decreased to 68%, or even 57% when only t-MRI was available. The sensitivity of HR-ESI/i-MRI compared favorably with those of MRI (76%), PET (69%) and ictal/interictal SPECT (64%).Interpretation: This study on a large patient group shows excellent sensitivity and specificity of ESI if 128 EEG channels or more are used for ESI and if the results are co-registered to the patient's individual MRI. Localization precision is as high as or even higher than established brain imaging techniques, providing excellent costeffectiveness in epilepsy evaluation. HR-ESI appears to be a valuable additional imaging tool, given that larger electrode arrays are easily and rapidly applied with modern EEG equipment and that structural MRI is nearly always available for these patients.

Acidez potencial estimada pelo pH SMP em solos do estado de Pernambuco

A determinação da acidez potencial (H + Al) extraída com a solução de acetato de cálcio 0,5 mol L-1 tem sido rotineiramente utilizada em laboratórios de análise de solo do Brasil. No entanto, por suas facilidades analíticas, têm-se estimado seus valores, utilizando o pH de equilíbrio da suspensão de solo com a solução SMP. Este trabalho objetivou estudar a relação entre pH SMP e H + Al em solos de Pernambuco, visando estabelecer uma equação que possa ser utilizada na estimativa da acidez potencial. O trabalho, realizado no laboratório de fertilidade do solo da Universidade Federal Rural de Pernambuco, utilizou 145 amostras de solo das várias regiões do Estado. Os resultados permitiram concluir que a equação H + Al = 0,4837 SMP² - 8,4855 SMP + 38,448 (R² = 0,90), expressando os valores de H + Al em cmol c dm-3, pode ser utilizada para estimar a acidez potencial pelo uso do método do pH SMP.

Funções de pedotransferência para predição da umidade retida a potenciais específicos em solos do estado de Pernambuco

Funções de pedotransferência são equações usadas para estimar características edáficas de difícil determinação a partir de outras mais facilmente obtidas. Apesar do bom número de equações disponíveis para estimativa da umidade retida a potenciais matriciais específicos, elas não devem ser usadas indiscriminadamente, pois, em sua maioria, foram desenvolvidas com solos de clima temperado e a partir de dados gerados por métodos diversos dos em uso nos laboratórios brasileiros. O presente trabalho teve por objetivos: (a) elaborar funções de pedotransferência para estimar o conteúdo de água nos potenciais de -33 e -1.500 kPa e a água disponível, a partir de dados granulométricos e de densidade do solo, para solos do estado de Pernambuco e (b) comparar a eficiência de predição das equações propostas em relação a equações similares, disponíveis na literatura. No desenvolvimento das equações, foi utilizada uma base de dados composta por 98 perfis de solos e 467 horizontes. Os perfis foram agrupados, de acordo com Sistema Brasileiro de Classificação de Solos, em 27 classes de terceiro nível categórico (grande grupo). As equações desenvolvidas apresentaram bons coeficientes de determinação e baixo erro de predição. A sistematização dos dados por atividade da fração argila ou grau aproximado de desenvolvimento ou classe textural não produziu melhorias na capacidade preditiva das pedofunções. As equações propostas em outros trabalhos apresentaram elevado erro de predição, o que restringe a sua utilização para solos do estado de Pernambuco.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.