Participación de rafts en enfermedades neurológicas / Role of rafts in neurological disorders

Introducción: Los rafts constituyen nano-dominios estructurales de naturaleza lipoproteica que propician la eficiente transducción de señales y la modulación de procesos fisiológicos asociados a la membrana plasmática. En el sistema nervioso, la alteración de estos dominios se ha asociado con el desarrollo de diversos padecimientos. Desarrollo: En el presente artículo se revisa el concepto de rafts, los procesos del sistema nervioso en los cuales están involucrados y su papel en distintas afectaciones, entre las que se destacan las enfermedades de Parkinson, Alzheimer y Huntington. Conclusiones: Dadas las evidencias de su participación en diversas neuropatologías, la preservación y/o reconstitución de los rafts se vislumbran como una atractiva estrategia terapéutica.(AU)

Introduction: Rafts are function-structural cell membrane nano-domains. They contribute to explain the efficiency of signal transduction at the low physiological membrane concentrations of the signaling partners by their clustering inside specialized signaling domains. Development: In this article, we review the current model of the membrane rafts and their physio-pathological relevance in the nervous system, including their role in Parkinson, Alzheimer, and Huntington diseases. Conclusions: Rafts disruption/dysfunction has been shown to relate diverse neurological diseases. Therefore, it has been suggested that preservation of membrane rafts may represent a strategy to prevent or delay neuronal dysfunctions in several diseases.(AU)

Role of rafts in neurological disorders. / Participación de rafts en enfermedades neurológicas.

INTRODUCTION: Rafts are function-structural cell membrane nano-domains. They contribute to explain the efficiency of signal transduction at the low physiological membrane concentrations of the signaling partners by their clustering inside specialized signaling domains. DEVELOPMENT: In this article, we review the current model of the membrane rafts and their physio-pathological relevance in the nervous system, including their role in Parkinson, Alzheimer, and Huntington diseases. CONCLUSIONS: Rafts disruption/dysfunction has been shown to relate diverse neurological diseases. Therefore, it has been suggested that preservation of membrane rafts may represent a strategy to prevent or delay neuronal dysfunctions in several diseases.

Óleo de peixe associado ao ácido ascórbico no diluidor para criopreservação de sêmen caprino / Fish oil associated to ascorbic acid in diluter for cryopreservation goat semen

O presente estudo teve como objetivo avaliar o efeito da inclusão de óleo de peixe associado ao ácido ascórbico no diluidor para criopreservação de sêmen caprino. Dois machos da raça Boer foram submetidos à coleta de sêmen pelo método de vagina artificial, sendo os ejaculados avaliados quanto aos aspectos físicos e morfológicos. Após avaliação, formou-se um pool, seguido do fracionamento em cinco grupos: G1 - diluidor citrato-gema e G2, G3, G4 e G5 - diluidor citrato-gema acrescido de 1,0; 2,0; 3,0 e 4,0% de óleo de peixe e 0,05% de ácido ascórbico, respectivamente. Após descongelamento, foram realizadas avaliações físicas do sêmen e os testes complementares de termorresistência lento (TTR), hiposmótico (HO), integridade acrossomal e compactação da cromatina espermática. Houve comportamento linear crescente (P<0,05) para motilidade pós-descongelamento. Não houve diferença (P>0,05) para vigor pós-descongelamento (2,00±0,24). No TTR não houve diferença (P>0,05) para motilidade e vigor espermáticos entre os tempos cinco e 180min, com médias inicial e final de 62,17±12,13 e 14,29±10,55 para motilidade e de 2,00±0,52 e 0,49±0,44 para vigor. Não houve diferença (P>0,05) para o HO, com porcentagem média de espermatozoides reativos de 23,5±5,96%. Houve comportamento linear crescente para acrossoma íntegro e decrescente para acrossoma irregular (P<0,05). Não houve diferença (P>0,05) na compactação da cromatina, com 97,06±1,17% de cromatina íntegra. A inclusão até 4% de óleo de peixe acrescido de ácido ascórbico no diluidor melhorou motilidade e integridade de acrossoma após a criopreservação.(AU)

The study aimed to evaluate the effect of fish oil inclusion associated with ascorbic acid in the thinner for goat semen cryopreservation. Two male Boers underwent semen collection through the artificial vagina method, ejaculates being then assessed for physical and morphological aspects. After evaluation, a pool was formed, followed by the split into five groups: G1 - yolk-citrate extender and G2, G3, G4 and G5 - yolk-citrate extender plus 1.0; 2.0; 3.0 and 4.0% fish oil and 0.05% ascorbic acid, respectively. After thawing, physical evaluations of semen were assessed and additional testing slow heat resistance (TTR), hiposmotic (HO), acrosome integrity and compression of sperm chromatin. There was linear increase (P<0.05) post-thaw motility. No difference was obtained for post-thaw vigor and there was no influence of the association of fish oil and ascorbic acid in TTR. Plasma membrane integrity, by hyposmotic test (HO), presented a mean of reactive spermatozoa of 23.5±5.96% (P>0.05). There was linear increase for intact acrosome and decreasing acrosome irregular (P<0.05). In the analysis of the chromatin compaction, approximately 3% of damages (P>0.05) were observed. The inclusion of 4% fish oil plus ascorbic acid in diluter improved motility and acrosome integrity after cryopreservation.(AU)

[Interaction of FABP4 with plasma membrane proteins of endothelial cells]. / Interacción de FABP4 con proteínas de membrana de células endoteliales.

INTRODUCTION: Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. METHODOLOGY: HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. RESULTS: The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. DISCUSSION: The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular.

Influencia de las acuaporinas en la génesis de múltiples enfermedades / Aquaporins Influence in the Genesis of Multiple Diseases

Las acuaporinas son una familia de proteínas que forman los canales de agua de la membrana. Están involucradas en una gran variedad de funciones fisiológicas y enfermedades humanas como el glaucoma, cáncer, epilepsia, obesidad entre otras. El descubrimiento de las acuaporinas cambia el panorama con respecto a la comprensión del transporte de agua en las membranas biológicas y a la naturaleza de las proteínas transportadoras en general, porque su estudio demuestra que existen canales en las membranas no sólo permeables a iones y que los movimientos de agua a través de las membranas son regulados por la célula en forma muy diferente a como se pensaba hace una década, esto implica comprender mejor los mecanismos fisiopatológicos que lleven al desarrollo de nuevas medidas terapéuticas y más eficaces para el tratamiento de las enfermedades.

Aquaporins are a family of membrane water channels. They are involved in a great variety of physiological functions and human diseases including, glaucoma, cancer, epilepsy, obesity among others. The discovery of the aquaporins have changed the view regarding the understanding of the transportation of water in the biological membranes and to the nature of the transporting proteins in general, because its study has showed that there are canals not only in permeable membranes to ions and the fact that the movements of water through the membranes are regulated by the cell in very different way, comparing to the considerations that existed a decade ago, this allows the understanding of physiopathologic mechanisms that lead to the development of therapeutic new measures better and more effective for the diseases treatment.

Influencia de las acuaporinas en la génesis de múltiples enfermedades / Aquaporins Influence in the Genesis of Multiple Diseases

Las acuaporinas son una familia de proteínas que forman los canales de agua de la membrana. Están involucradas en una gran variedad de funciones fisiológicas y enfermedades humanas como el glaucoma, cáncer, epilepsia, obesidad entre otras. El descubrimiento de las acuaporinas cambia el panorama con respecto a la comprensión del transporte de agua en las membranas biológicas y a la naturaleza de las proteínas transportadoras en general, porque su estudio demuestra que existen canales en las membranas no sólo permeables a iones y que los movimientos de agua a través de las membranas son regulados por la célula en forma muy diferente a como se pensaba hace una década, esto implica comprender mejor los mecanismos fisiopatológicos que lleven al desarrollo de nuevas medidas terapéuticas y más eficaces para el tratamiento de las enfermedades.(AU)

Aquaporins are a family of membrane water channels. They are involved in a great variety of physiological functions and human diseases including, glaucoma, cancer, epilepsy, obesity among others. The discovery of the aquaporins have changed the view regarding the understanding of the transportation of water in the biological membranes and to the nature of the transporting proteins in general, because its study has showed that there are canals not only in permeable membranes to ions and the fact that the movements of water through the membranes are regulated by the cell in very different way, comparing to the considerations that existed a decade ago, this allows the understanding of physiopathologic mechanisms that lead to the development of therapeutic new measures better and more effective for the diseases treatment.(AU)

Modelagem molecular aplicada à elucidação dos mecanismos envolvidos na ação antiproliferativa e hemolítica das alquilfosfocolinas / Molecular modeling applied to the elucidation of the antiproliferative and hemolytic mechanisms of action of alkylphosphocholines

As alquilfosfocolinas (APC) são uma classe de fármacos derivados de fosfolipídios endógenos que apresentam potencial antitumoral. Diferentemente de outros fármacos antitumorais que agem no DNA da célula, as APCs têm como primeiro local de ação a membrana plasmática e proteínas de sinalização, como a PKC. O objetivo desse trabalho é elucidar, através de metodologias computacionais, os possíveis mecanismos de ação das APCs que provocam hemólise, inibição da PKC e interação com membranas celulares. Inicialmente, a toxicidade de um conjunto de 34 APCs foi estudada pelos métodos quimiométricos de Análise de Agrupamentos Hierárquicos (HCA) e Componentes Principais (PCA). As moléculas foram simuladas com dinâmica molecular (DM) e propriedades físico-químicas e estruturais foram calculadas para os confôrmeros de menor energia. Após aplicação de HCA e PCA, as APCs foram divididas em 3 grupos, de acordo com suas características estruturais. Os resultados sugerem que a presença de grupos catiônicos volumosos, ou anéis como adamantila e ciclohexila, aumentam a hemólise de compostos de cadeia alquílica longa. Anéis macrocíclicos como ciclopentadecila parecem ser importantes para o potencial hemolítico de compostos com cadeia alquílica curta. Com relação a compostos sem anéis e de cadeia linear, grupos catiônicos menos volumosos parecem favorecer a hemólise. Na próxima etapa do estudo, 7 derivados de APC, com diferentes grupos catiônicos, foram selecionados e ancorados no domínio C2 da PKCα. O intuito foi mapear resíduos de aminoácidos importantes para a interação dos ligantes com a enzima, e comparar com o modo de ligação do ativador endógeno fosfatidilserina (PS). Mais uma vez, HCA e PCA foram aplicados para extrair informação relevante do mapeamento. Os resultados mostraram que as cadeias laterais de Pro188, Asn189, Arg216, Trp247, Asp249 e Thr250 não permitem a aproximação adequada do ligante, o que impede que a porção fosforila se coordene com um dos átomos de cálcio. A porção catiônica da PS, em contrapartida, consegue estabelecer ligação-hidrogênio com Asn189 de forma a posicionar os oxigênios da fosforila para interagir, ao mesmo tempo, com o átomo de cálcio. Com menos pontos de coordenação, a afinidade de ligação do cálcio pela PKCα diminui e a ativação da enzima fica comprometida, interrompendo toda a cascata de sinalização que depende dela. A parte final desse trabalho se dedicou ao estudo da interação da miltefosina com diferentes bicamadas lipídicas sob o ponto de vista termodinâmico. Oito bicamadas de diferentes fosfolipídios foram simuladas por DM e a interação energética da miltefosina foi calculada por Umbrella Sampling. Os resultados mostraram que a miltefosina apresenta maior partição em bicamadas contendo colesterol, sendo a miscibilidade nesses sistemas cerca de 76 vezes maior que os valores encontrados para bicamadas sem colesterol. Além disso, verificou-se que a internalização da miltefosina é mais fácil em regiões contendo lipídeos poli-insaturados, provavelmente devido ao empacotamento mais frouxo da bicamada. Os dados sugerem que a miltefosina age principalmente em rafts lipídicos e que células contendo mais lipídicos poli-insaturados podem incorporar maior quantidade do fármaco

Alquilfosfocolines (APCs) comprise a class of drugs with antitumor activity derived from endogenous phospholipids. Differently from other drugs whose primary site of action is the DNA, APCs act firstly in the plasma membrane and signaling proteins, such as PKC. The main objective of this work is to elucidate, via computational approaches, the possible mechanisms of actions that cause hemolysis, PKC inhibition and interaction with cellular membranes. Initially, a set of 34 APCs was studied by means of Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA). The molecules were simulated by means of molecular dynamics simulations (MD) and molecular and structural properties were calculated for the lowest-energy conformer. After HCA and PCA methodologies, the set was divided into 3 groups according to their structural features. The findings suggest that the presence of bulky cationic moieties, or the adamantyl and cyclohexil rings, increase the hemolytic potential of compounds with long alkyl chains. Macrocyclic rings, such as cyclopentadecyl, seem to be important to elevate the hemolysis of compounds with short alkyl chains. Regarding linear carbon chain derivatives with no ring substitution, less bulky cationic head groups seem to favor hemolysis. In the next step of this work, 7 APC derivatives were selected and docked in the C2 domain of PKCα. The aim now was to map the residues relevant for ligands interaction compared to the binding mode of the endogenous activator, phosphatidylserine (PS). HCA and PCA were again applied in order to extract relevant information from the mapping. The results showed that the lateral chains of Pro188, Asn189, Arg216, Trp247, Asp249 and Thr250 do not allow the proper approximation of the ligands, impeding the phosphoryl moiety from coordinating with one of the calcium atoms. On the other hand, the cationic moiety of PS forms hydrogen-bonding with Asn189 in order to position the oxygens to interact, at the same time, with a calcium atom. With less coordination sites, calcium binding affinity diminishes and the enzyme activation is compromised, interrupting the signaling cascade. The final part of this work was dedicated to the study of miltefosine interaction with different lipid bilayers from the thermodynamics standpoint. Eight bilayers were simulated with MD and the energetic interaction was calculated via Umbrella Sampling simulations. The findings showed that miltefosine has higher partition in bilayers containing cholesterol, with miscibility of about 76 times higher than the values referring to bilayers without cholesterol. Moreover, it was observed that the internalization of miltefosine is facilitated in regions containing polyunsaturated lipids, probably due to the looser packing. The data suggest that miltefosine acts primarily in lipid rafts, and that cells containing more polyunsaturated lipids in their membranes can incorporate higher quantities of this drug