ABSTRACT
Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.
Subject(s)
Protein Engineering/methods , Recombinant Fusion Proteins/chemistry , Tissue Plasminogen Activator/chemistry , Animals , CHO Cells , Cloning, Molecular , Computer Simulation , Cricetinae , Cricetulus , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Fibrin/metabolism , Fibrinogen/metabolism , Kringles , Models, Molecular , Plasminogen/metabolism , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tissue Plasminogen Activator/biosynthesis , Tissue Plasminogen Activator/genetics , Tissue Plasminogen Activator/metabolismABSTRACT
BACKGROUND: D2 dopamine receptor gene has been reported to be one of the most relevant candidate genes in schizophrenia. In this study, we investigated the association between TaqIA and TaqIB dopamine D2 receptor polymorphisms and psychopathology of schizophrenia. METHODS: The study subjects were 38 acutely exacerbated schizophrenic patients who were all Iranian descent. The control population consisted of 63 healthy individuals with almost the same age as patients and were also of Iranian decent. The TaqIA and TaqIB genotypes, the A1 and A2 alleles, and the B1 and B2 were determined by restriction fragment length polymorphism of the amplified DNA fragments by polymerase chain reaction . RESULTS: For each polymorphism (A or B) the patients were categorized according to their genotype into three groups; i.e. the patients with alleles A1/A1, A1/A2, A2/A2; B1/B1, B1/B2, and B2/B2. No significant association was found between Taq1A or Taq1B gene polymorphisms and schizophrenia in patients compared to the controls. When study subjects were stratified according to their gender, the distribution of the A1/A1 genotype did was significantly different in both men and women (patients vs. controls). CONCLUSION: Our findings show that there is no genetic association between Taq1A and Taq1B gene polymorphisms and schizophrenia. Further clinical studies should be conducted to confirm and further evaluate these findings.
