RESUMO
HIV is a public health problem, which makes necessary the development of new drugs. Natural products are known for their anti-HIV potential and a good strategy to suggest its mechanism of action is using in silico tools. Herein, diterpenes 1-3 had the binding mode evaluated in the HIV-1 glycoprotein; and properties ADMET in silico performed. In molecular docking important interactions between the hydrophobic cavity, and 1 and 2 were observed. In the molecular dynamics, 1 remained stable covering the entire hydrophobic cavity and performed hydrogen bond during all simulation. ADMET evaluation showed good properties for the diterpenes. Based on these findings, it was possible to suggest the potential from natural products as entry inhibitor and HIV-1 treatment.
Assuntos
Produtos Biológicos , Diterpenos , HIV-1 , Phaeophyceae , Simulação de Acoplamento Molecular , Phaeophyceae/química , Diterpenos/químicaRESUMO
KEY MESSAGE: NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. In this work, we used molecular modeling approach and site-directed mutagenesis to characterized the NADP-ME2 structural determinants necessary for allosteric regulation providing new insights for enzyme optimization. Structure-function studies contribute to deciphering how small modifications in the primary structure could introduce desirable characteristics into enzymes without affecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from different species facilitates comparisons between sequence and structure. Specifically, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also fit into fumarate's pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identified. The substitution of one of those residues, L62, by a less flexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate affinities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.
Assuntos
Aminoácidos/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Malato Desidrogenase (NADP+)/química , Malato Desidrogenase (NADP+)/metabolismo , Transdução de Sinais , Sítio Alostérico , Fluorescência , Cinética , Simulação de Acoplamento Molecular , Proteínas Mutantes/metabolismo , Mutação/genéticaRESUMO
Brain Expressed X-linked (BEX) protein family consists of five members in humans and is highly expressed during neuronal development. They are known to participate in cell cycle and in signaling pathways involved in neurodegeneration and cancer. BEX3 possess a conserved leucine-rich nuclear export signal and experimental data confirmed BEX3 nucleocytoplasmic shuttling. Previous data revealed that mouse BEX3 auto-associates in an oligomer rich in intrinsic disorder. In this work, we show that human BEX3 (hBEX3) has well-defined three-dimensional structure in the presence of small fragments of tRNA (tRFs). Conversely, the nucleic acids-free purified hBEX3 presented disordered structure. Small-angle X-ray scattering data revealed that in the presence of tRFs, hBEX3 adopts compact globular fold, which is very distinct from the elongated high-order oligomer formed by the pure protein. Furthermore, microscopy showed that hBEX3 undergoes condensation in micron-sized protein-rich droplets in vitro. In the presence of tRFs, biomolecular condensates were smaller and in higher number, showing acridine orange green fluorescence emission, which corroborated with the presence of base-paired nucleic acids. Additionally, we found that over time hBEX3 transits from liquid condensates to aggregates that are reversible upon temperature increment and dissolved by 1,6-hexanediol. hBEX3 assemblies display different morphology in the presence of the tRFs that seems to protect from amyloid formation. Collectively, our findings support a role for tRFs in hBEX3 disorder-to-order transition and modulation of phase transitions. Moreover, hBEX3 aggregation-prone features and the specificity in interaction with tRNA fragments advocate paramount importance toward understanding BEX family involvement in neurodevelopment and cell death.
Assuntos
Proteínas Reguladoras de Apoptose/química , Regulação da Expressão Gênica , RNA Bacteriano/genética , RNA de Transferência/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Escherichia coli/genética , Humanos , Transição de Fase , Conformação Proteica , RNA Bacteriano/química , RNA de Transferência/químicaRESUMO
Our understanding of the phylogenetic and structural characteristics of the Merkel Cell Polyomavirus (MCPyV) is increasing but still scarce, especially in samples originating from South America. In order to investigate the properties of MCPyV circulating in the continent in more detail, MCPyV Viral Protein 1 (VP1) sequences from five basal cell carcinoma (BCC) and four saliva samples from Brazilian individuals were evaluated from the phylogenetic and structural standpoint, along with all complete MCPyV VP1 sequences available at Genbank database so far. The VP1 phylogenetic analysis confirmed the previously reported pattern of geographic distribution of MCPyV genotypes and the complexity of the South-American clade. The nine Brazilian samples were equally distributed in the South-American (3 saliva samples); North American/European (2 BCC and 1 saliva sample); and in the African clades (3 BCC). The classification of mutations according to the functional regions of VP1 protein revealed a differentiated pattern for South-American sequences, with higher number of mutations on the neutralizing epitope loops and lower on the region of C-terminus, responsible for capsid formation, when compared to other continents. In conclusion, the phylogenetic analysis showed that the distribution of Brazilian VP1 sequences agrees with the ethnic composition of the country, indicating that VP1 can be successfully used for MCPyV phylogenetic studies. Finally, the structural analysis suggests that some mutations could have impact on the protein folding, membrane binding or antibody escape, and therefore they should be further studied.
Assuntos
Proteínas do Capsídeo/genética , Variação Genética , Poliomavírus das Células de Merkel/classificação , Poliomavírus das Células de Merkel/isolamento & purificação , Filogenia , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Brasil , Carcinoma Basocelular/virologia , Epitopos de Linfócito B/genética , Poliomavírus das Células de Merkel/genética , Mutação de Sentido IncorretoRESUMO
Polyomavirus BK (BKPyV) T-antigens (large and small tumor antigens, or Lt-ag and st-ag, respectively), control key aspects of viral replication and are able to regulate cell cycle, promoting cell proliferation. However, the structural effects of genetic mutations on T-antigens are poorly investigated. In this study, 214 sequences of T-antigens from individuals with different BKPyV infections (16 renal transplant with nephropathy; 78 asymptomatic renal transplant; 24 hematopoietic stem cell transplant with hemorrhagic cystitis; 96 healthy non-transplant), were analyzed from the genetic and structural standpoints. We found a high concentration of non-synonymous mutations at inter-domains and hexamerization regions of both proteins, being five of them under positive selection in the Lt-ag but none in the st-ag. The in silico analysis indicated that two mutations, located at positions 164 in the st-ag and 592 in the Lt-ag, would significantly affect the interaction with PP2A and p53 cell targets, respectively, although they were not associated to a specific clinical status. No mutations were detected on the J-domains or at the ATPase motif. In sum, the profile of the mutations found seem not to be associated to increased morbidity. This is the first work to analyze structural modifications on T-antigens in different BKPyV infections, and managed to map conserved and variable regions of the T-antigens, which will be helpful for the study of new antiviral drugs.
Assuntos
Antígenos Virais de Tumores/genética , Vírus BK/classificação , Vírus BK/genética , Variação Genética , Mutação de Sentido Incorreto , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Vírus BK/isolamento & purificação , DNA Viral/química , DNA Viral/genética , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Análise de Sequência de DNARESUMO
Merkel cell polyomavirus (MCPyV) large T antigen (LT-ag) is frequently found truncated in Merkel cell carcinomas (MCC) and it is considered a major tumor-specific signature. Nonetheless, the biological role of LT-ag nontruncated mutations is largely unknown. In this study, MCPyV LT-ag second exon from 11 non-MCC oral samples and NCBI sequences derived from different anatomical sites were studied from the genetic and structural standpoint. As expected, the LT-ag mutation profile was influenced by the geographical origin of the sample, although nonsynonymous mutations were more frequent in lesional tissues. Our in silico study suggests that the mutations found would not significantly affect protein functions, regardless of sample category. This work presents a thorough investigation of the structural and functional properties of LT-ag nontruncated mutations in MCPyV. Our results sustain the geographical influence of the MCPyV genetic profile, but do not discard genetic tissue specificities. Further investigation involving other genetic segments in healthy and lesional tissues are necessary to improve our knowledge on MCPyV pathogenesis.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/imunologia , Carcinoma de Célula de Merkel/virologia , Poliomavírus das Células de Merkel/imunologia , Neoplasias Cutâneas/virologia , Sequência de Bases , Simulação por Computador , Éxons/genética , Humanos , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Estrutura Terciária de Proteína , Saliva/virologia , Análise de Sequência de DNA , Pele/virologiaRESUMO
La muerte súbita es la situación de máxima urgencia médica. Muchas de estas muertes se producen en la vía pública en presencia de otras personas. Se ha comprobado repetidamente que, en esta situación, el porcentaje de fallecimientos supera el 90% y que entre los supervivientes más de la mitad presentará algún tipo de secuela neurológica. Todo ello indica que, teóricamente, se podrían recuperar muchos de estos enfermos si se inicia en maniobras de reanimación cardiopulmonar (RCP) básica por parte del primer interviniente. Sin embargo, esto sucede en menos del 25% de los paros cardiorrespiratorios (PCR) presenciados por testigos. Por ello, deben implementarse cuantas medidas conduzcan a un incremento del conocimiento de las maniobras de RCP básica a la población general. Numerosas instituciones y sociedades médicas internacionales han recomendado que la enseñanza de la RCP básica se introduzca durante la educación obligatoria, ya que la escuela constituye un ámbito ideal para iniciar a la población en el conocimiento y el aprendizaje de las técnicas básicas que forman parte de la RCP y todos los ciudadanos pasan por esta fase educativa. Reconociendo esta importancia, se ha enviado un proyecto de ley a la Cámara de Diputados para incorporar la enseñanza de técnicas de RCP al currículo escolar del nivel secundario en todas las instituciones de educación pública del país, sean de gestión estatal, privada, cooperativa o social. Existen numerosas experiencias en este sentido, que han obtenido resultados, implantación y pervivencia diversos. Este trabajo analiza la experiencia de los autores adquirida durante el desarrollo del PROCES, un programa dirigido a los estudiantes de 15 y 16 años que se lleva a cabo desde hace 10 años en la ciudad de Barcelona...
Assuntos
Humanos , Educação , Reanimação Cardiopulmonar , Estudantes , Morte , Morte Súbita , EmergênciasRESUMO
El desarrollo tecnológico requiere de métodos analíticos confiables que permitan la cuantificación del fármaco en diferentes etapas de la investigación. El objetivo de este trabajo fue las validaciones prospectivas de 2 métodos analíticos, uno por UV-VIS y otro por cromatografía líquida de alta eficiencia, desarrolladas para el control del proceso de fabricación, de la calidad y para el estudio de estabilidad de ketamina. A las técnicas se le determinaron los parámetros de desempeño, especificidad, linealidad, exactitud, rango y precisión. Los resultados alcanzados permiten concluir que ambos métodos cumplen con los requisitos establecidos como aceptables para cada uno de los casos en el rango de concentraciones establecido. El método espectrofotométrico puede utilizarse en el control del proceso de producción y el control de calidad del producto terminado al igual que el método cromatográfico; el primero, es de elección para el control de proceso de fabricación por su sencillez y rapidez, y el segundo, para el estudio de estabilidad del producto por su elevada especificidad.
Technological development requires of reliable and analytical methods to quantify drugs in different stages of research. Aim of present paper was the prospective validations of two analytical methods, one by UV-VIS, and the other by high performance liquid chromatography, developed to control of manufacture process, quality, and to study of Ketamine stability. Techniques included the following parameters: performance, specificity, linearity, accuracy, rank, and precision. Results achieved allow concluding that both methods fulfill the criteria established as fair to each of the cases in rank of concentrations stetted. Spectrophotometry method may be used in control of production process and in control of quality of end product just like the chromatography method, the first one, is the choice to control of manufacture process due to its simplicity and speed, and the second one, to study of product stability due to its high specificity.
Assuntos
Anestésicos Dissociativos/análise , Controle de Qualidade , Estudos de Validação como Assunto , Cromatografia Líquida de Alta Pressão/métodos , Espectrofotometria/métodosRESUMO
Se desarrolló una metodología de limpieza para aplicar en áreas de producción y desarrollo donde se trabaje con penicilamina, un compuesto altamente contaminante y tóxico. Se validó un método por cromatografía líquida de alta eficiencia que fue seleccionado por la versatilidad de este tipo de técnicas para la detección de trazas. El método resultó específico, lineal, exacto y preciso en el rango de concentraciones de trabajo. Se determinaron los límites de detección y cuantificación. Se utilizó el método de muestreo por hisopado, con el que se obtuvieron resultados satisfactorios y se concluyó que los procedimientos de limpieza resultaron efectivos para el peor de los escenarios
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , PenicilaminaRESUMO
El reasec es un antidiarreico cuyo efecto viene dado por la asociación de 2 principios activos, atropina sulfato y difenoxilato clorhidrato. La unión de ambos trae como resultado la inhibición del peristaltismo del tracto gastrointestinal que puede ser posible tanto a nivel central como local. De la literatura revisada para el caso del difenoxilato clorhidrato se escogió el método por cromatografía líquida de alta eficiencia por aprovechar la posibilidad de que se trataba del método propuesto en el registro de medicamentos para el estudio de estabilidad en la formulación de este principio activo. El método para la determinación de atropina sulfato se encuentra reportado en la USP 23 y el criterio de selección fue uno de los menos complejos en la cuantificación de esta. Teniendo en cuenta las regulaciones establecidas que aseguran el cumplimiento de las buenas prácticas de producción, el presente trabajo se propone la validación prospectiva de los métodos para la cuantificación de los principios activos componentes del reasec, por lo que se realizaron los estudios de especificidad, exactitud, precisión, linealidad y rango. En ambos casos se cumplieron con los requisitos establecidos a los métodos analíticos que se encuentran dentro de la categoría I por ser empleados para la cuantificación de los componentes activos de la formulación. Los resultados obtenidos demostraron que ambos métodos analíticos son fiables por permitir la cuantificación de los 2 principios activos y cumplir además, con los requisitos establecidos para los parámetros evaluados dentro de la categoría a la que pertenecen cada uno