Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)

Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.

Síndrome metabólica: um estado pró-trombótico / Metabolic syndrome: a prothrombotic state

Dentre os vários componentes considerados na definição da síndrome metabólica encontra-se o estado pró-trombótico. Esse estado pró-trombótico é caracterizado pelo desequilíbrio entre fatores pró-coagulantes e pró-fibrinolíticos. Clinicamente o estado pró-trombótico da síndrome metabólica pode ser caracterizado pela elevação do fibrinogênio e do PAI-1 e pela ativação de vias de coagulação. Além da ativação de vias de coagulação e do aumento de fatores que desencadeiam fenômenostrombóticos no paciente com a síndrome metabólica, o agrupamento de fatores de risco cardiovascular encontrado nesses pacientes está associado também com a disfunção endotelial. O endotélio, como já se sabe, regula funções tais como controle do tônus vascular, metabolismo de lipídios, equilíbrio fibrinólise-trombose, agregação plaquetária, resposta inflamatória (permeabilidade celular) e proliferação celular vascular. De modo que ele assume papel muito importante como elo fisiopatológico desde a patogênese da aterosclerose incipiente até as complicações mais graves. Essa situação é preocupante pelas evidências epidemiológicas, que demonstram risco de eventos cardiovasculares aterotrombóticos aumentado associado coma síndrome metabólica.

Plasminogen activator inhibtior-1 and fibrinogen in ischemic heart disease

Three groups were included in the study. Group I of 40 males with stable angina with mean age of 52.1 +/- 5.43 years. Group II of 40 male patients with acute myocardial infarction [MI] with a mean age of 51.2 +/- 5.7 years. Control group of 10 normal males with mean age of 50.8 +/- 8.04 years. The results showed significant elevation of serum cholesterol in groups I and II over control group [P <0.0001 - <0.05], respectively. Triglyceride levels were elevated nonsignificantly in group I [P >0.05] but showed highly significant elevation in group II over controls [P <0.0001]. Plasma fibrogen levels were significantly elevated in both groups over controls [P <0.0001]. Plasma PAI-1 showed a nonsignificant difference in group I [P >0.05] and a highly significant elevation in group II over control group [P <0.0001]. Patients with acute MI [group II] showed a significant rise of triglycerides and PAI-1 over those with stable angina [group I] [P <0.0001]. The results of this study showed that plasma fibrinogen is a significant risk factor in both stable angina and acute MI. They also support the hypothesis that there is a significant correlation between triglycerides and PAI-1 as important risk factors only in acute MI, while serum cholesterol is rather more important as atherogenic factor