A general model for the conformational switch in 5S RNA during protein synthesis.
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Biomedical subjects
P Jagadeeswaran
Publications and source records attributed to P Jagadeeswaran.
Analysis of blood coagulation in the zebrafish.
The zebrafish (Danio rerio) is a unique animal model in which saturation mutagenesis has been used to identify genes involved in vertebrate development. The relevance of the zebrafish as a genetic model for hemostasis depends, in large part, on the degree of similarity between the zebrafish and mammalian systems. The diminutive size of the zebrafish poses technical problems for analysis of coagulation. This study describes methods to obtain citrated whole blood and plasma from the zebrafish, analyze in vitro coagulation in small plasma volumes, obtain uniform dosing of zebrafish with oral anticoagulants, and demonstrate specific factor activities via chromogenic assays. Analysis of the zebrafish system demonstrates the presence of both the intrinsic and extrinsic pathways of coagulation, evidence for prothrombin, factor X, protein C, antithrombin, and heparin cofactor II activity, and a requirement for vitamin K dependent gamma-carboxylation of zebrafish hemostatic proteins. Induction of a morphologically recognizable bleeding phenotype by warfarin treatment is also demonstrated. Characterization of zebrafish coagulation provides evidence that major hemostatic pathways are conserved between zebrafish and man. These similarities indicate that the zebrafish is a relevant genetic model for identification of novel genes involved in hemostasis and thrombosis.
Animals↗
Developmental expression of vitamin K-dependent gamma-carboxylase activity in zebrafish embryos: effect of warfarin.
Vitamin K-dependent gamma-carboxylation is an essential posttranslational modification required for the functional activity of coagulation proteins such as factors VII, IX, X, and prothrombin. Warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, was used in earlier work on adult zebrafish to provide evidence for the presence of vitamin K-dependent carboxylase in zebrafish. Here we demonstrate the presence of vitamin K-dependent carboxylase activity in zebrafish by directly assaying the microsomal fraction prepared from adult, unfertilized eggs, and embryos from different developmental stages. Gamma-carboxylase activity was detected both before and after fertilization of embryos and the activity levels remained relatively constant from 6 h postfertilization (hpf) through other advanced stages of development. The expression of activity in the early embryos (0-6 hpf) may be due to the presence of maternal protein since the activity was detected even in the unfertilized eggs. Gamma-carboxylase activity in the eggs as well as early embryos suggested that vitamin K-dependent carboxylase is important throughout development. The detection of vitamin K-dependent carboxylase mRNA by RT-PCR and inhibitor studies using warfarin confirmed these activity results. Further, these studies provide a basis for selecting warfarin-resistant zebrafish mutants in order to find genes regulating gamma-carboxylase activity including the yet unidentified vitamin K-epoxide reductase.
Amino Acid Sequence↗