ABSTRACT
The dengue virus (DENV) is the causative agent of the viral infection dengue fever. In spite of all the efforts made to prevent the spread of the disease, once it is contracted, there is no specific treatment for dengue and the WHO guidelines are limited to rest and symptomatic treatment. In its reproductive cycle, DENV utilizes the NS2B-NS3pro, a serine protease, to cleave the viral polyprotein into its constituents. This enzyme is essential for the virus lifecycle, and presents an attractive target for the development of specific dengue treatments. Here we used a hybrid Quantum Mechanics and Molecular Mechanics (QM/MM) Molecular Dynamics approach and Umbrella Sampling to study the first step (acylation) of the reaction catalyzed by NS2B-NS3pro, using the Pairwise Distance Directed Gaussian PM3 (PDDG/PM3) semi-empirical Hamiltonian for the QM subsystem, and Amber ff99SB for the MM subsystem. Our results indicate that the nucleophilic attack on the substrate by Ser135 occurs in a stepwise manner, in which a proton transfer to His51 first activates Ser135, which only later attacks the substrate. The rate-determining step is the Ser135 activation, with a barrier of 24.1 kcal mol-1. Water molecules completing the oxyanion hole stabilize the negative charge formed on the carbonyl oxygen of the substrate. The final step in the process is a proton transfer from His51 to the substrate's nitrogen, which happens with a lower barrier of 5.1 kcal mol-1, and leads directly to the breakage of the peptide bond.
Subject(s)
Dengue Virus/enzymology , Molecular Dynamics Simulation , Serine Proteases , Viral Proteins , CatalysisABSTRACT
Human hemoglobin was isolated and purified by anion exchange chromatography. To isolate hemoglobin, outdated red blood cells (RBC) were transformed into carbonylhemoglobin, by reaction with carbon monoxide, and submitted to washing/centrifugation procedures, to eliminate other plasma proteins. Albumin was quantified in each supernatant, by the bromcresol green method. Hemolysis was performed in three different hypotonic media (water, 0.01 M NaCl and 5 mM Tris/HCl buffer at pH 7.4), at 8 degrees C for 24 h. Sonication for 5 min was also used to lyse RBC. After isolation of hemoglobin, additional purification was carried out by anion exchange chromatography on AG MP-1, Q-SFF and both exchangers. Hemoglobin concentration of hemolysates and of purified solutions were determined by the hemiglobincyanide method. Residual phospholipids were extracted from the four different hemolysates, as well as from the purified hemoglobin solutions, and were analyzed by high performance liquid chromatography. Native and SDS-polyacrylamide gel electrophoresis experiments were performed on purified hemoglobin samples to verify the presence of proteins other than hemoglobin. According to the results, the hemolysis conditions have influence on the purification of hemoglobin.
Subject(s)
Chemistry, Clinical/methods , Hemoglobins/chemistry , Hemoglobins/isolation & purification , Albumins/chemistry , Blood/metabolism , Centrifugation , Chromatography , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis , Electrophoresis, Polyacrylamide Gel , Erythrocytes , Hemolysis , Humans , Oxygen/metabolism , Phospholipids/chemistry , Time FactorsABSTRACT
A utilizaçäo terapêutica de doses elevadas de imunossupressores pode promover diversas complicaçöes, principalmente infecciosas. OBJETIVOS: Avaliar as complicaçöes secundárias ao uso de corticóide e ciclofosfamida em portadores de nefropatias. MÉTODOS: Foram estudados retrospectivamente 76 pacientes atendidos no Hospital das Clínicas da Faculdade de Medicina de Botucatu - UNESP, sendo divididos em três grupos: G1= Lúpus Eritematoso Sistêmico sem lesäo renal (n=15); G2= nefrite lúpica (n=33) e G3= síndrome nefrótica por glomerulopatia idiopática (n=28). RESULTADOS: Näo houve diferença em relaçäo ao tempo de acompanhamento (G1= 42,4 ± 51, G2= 52,3 ± 51, G3= 41,8 ± 47,8 meses), dose total de corticóide utilizada (G1= 20, G2= 28, G3= 16 gramas) e tempo de uso da droga (G1= 20, G2= 26, G3= 14,5 meses). Quanto ao uso de ciclofosfamida, näo houve diferença na percentagem de pacientes que a utilizaram (13 por cento no G1, 51 por cento no G2, 28 por cento no G3), porém pacientes do G1 receberam dose total menor que G2 (mediana de zero e um grama, respectivamente -- p<0.05). Aspecto cushingóide, manifestaçöes gástricas, distúrbios comportamentais, diabetes mellitus e alteraçöes oculares ocorreram nos três grupos, sem diferença estatística. Quanto às complicaçöes infecciosas, aquelas consideradas clinicamente mais graves, foram mais freqüentes no G2 (G1= 6 por cento, G2= 15 por cento, G3= 0 por cento - p<0.05), o mesmo ocorrendo em relaçäo aos óbitos (7 por cento no G1, 30 por cento no G2, 0 por cento no G3 -- p<0.05). CONCLUSÖES: Pacientes portadores de nefrite lúpica apresentaram maior freqüência de complicaçöes infecciosas decorrentes da imunossupressäo prolongada, o que pode representar um marcador de gravidade deste tipo de lesäo
