Peptides derived from plasma proteins released by bothropasin, a metalloprotease present in the Bothrops jararaca venom.

Viperid snake venoms contain proteases that affect hemostasis by degrading important proteins such as those that participate in the coagulation cascade. The Bothrops jararaca venom presents as its main components metallo and serine proteases, which comprise around 65% of the venom composition. Bothropasin is a hemorrhagic metalloprotease from the B. jararaca venom which causes disruption of the basement membrane of the vascular endothelium, resulting in bleeding. Although the bothropasin ability to degrade plasmatic and extracellular matrix proteins in vitro has been described, the primary sequence of the released peptides is unknown. This research study presents the peptide identification from both fibrinogen and fibronectin, generated by bothropasin proteolytic activity. Among the fibrinogen derived peptides identified by mass spectrometry, analogous of endogenous products like the fibrinopeptides A and B were found, as well as other sequences described in the literature with vasoactive or antiangiogenic properties. A series of peptides derived from fibronectin by the action of bothropasin were described, and for most of them no biological activity has been described. However, exceptionally a peptide that is known as a bond site for B cells was found. This study indicates that, beyond to the degradation of human proteins, bothropasin can generate bioactive peptides, which may participate in the envenoming process by Bothrops snakes. Also important, the knowledge of the formed peptides, based on the cleavage sites of the hydrolyzed proteins, provided the opportunity to study the primary specificity of bothropasin.

Estandarización del método de centrifugación en placa para el aislamiento del virus dengue / Rapid centrifugation assay standarization for dengue virus isolation

Se estandarizó el método de centrifugación en placa, para el aislamiento del virus dengue a partir de muestras de suero humano. Se utilizó la línea celular C6/36-HT determinándose los valores óptimos de velocidad de centrifugación, volumen de inóculo, dilución de suero y tiempo de incubación. Posteriormente, 22 muestras de suero con aislamiento viral positivo y cepas referenciales de los cuatro serotipos del virus dengue, fueron procesadas simultáneamente por el método de centrifugación en placa y el método convencional de cultivo en tubo, los aislamientos fueron tipificados mediante inmunofluorescencia indirecta empleando anticuerpos monoclonales. Se optimizó el método de centrifugación en placa inoculando 200 ul de dilución de suero 1/20, centrifugación a 1600 rpm/30 min, presentando sensibilidad de 95,5 por ciento a cinco días postinoculación. Se concluye que el método de centrifugación en placa mejora el porcentaje de aislamiento, con significativa reducción en tiempo de aislamiento del virus dengue.

The plate centrifugation assay was standardized for dengue virus isolation from serum samples. C6/36-HT cells were used determining the optimal values for centrifugation spin speed, inoculum, sera dilution, and incubation time. Then, 22 positive serum samples with viral isolation and viral strains of the four reference dengue virus serotypes were tested simultaneously by the standardized plate centrifugation method and the conventional tube culture. The isolations were typified by indirect immunofluorescent test using monoclonal antibodies. The plate centrifugation method was optimizedto 200 uL of inoculum, dilution of sera 1/20, centrifugation speed at 1600 rpm/30 min, and sensitivity of 95,5 per cent after 5 days post-inoculation. We concluded that the plate centrifugation method increased dengue virus isolation, with a significant reduction of the time of isolation for dengue virus.