Characterization of an Inhibitor of Neuronal Plasminogen Activator Released by Heart Cells

A basic understanding of growth cone dynamics and developmental events involving growth cones requires an understanding of the function and regulation of molecules associated with and released by growth cones. Rat sympathetic neurons in culture release a urokinase-like plasminogen activator from their distal processes and/or growth cones (Pittman, 1985a). When sympathetic neurons are grown in cocultures with heart cells, however, plasminogen activator activity is not detected. The absence of plasminogen activator activity in cocultures of sympathetic neurons and heart cells appears to be due to the release of an inhibitor of plasminogen activator by heart cells. This inhibitor has a molecular weight of approximately 50 kDa in the presence of SDS and apparent molecular weights of approximately 50 and greater than 2000 kDa under native conditions. A significant fraction of the large- molecular-weight form of the inhibitor is converted to the smaller form following treatment with heparinase. Extremely stable complexes of 68 and 80 kDa are formed between the heart inhibitor and the plasminogen activator, urokinase, such that the complexes withstand boiling in SDS/mercaptoethanol. The data are consistent with the formation of an 80 kDa urokinase-inhibitor complex in the presence of heparan sulfate proteoglycan and a 68 kDa complex in the absence of heparan sulfate proteoglycan. A highly purified preparation of the heart inhibitor produces a 2- to 3-fold increase in neurite outgrowth from sympathetic neurons. These data indicate that the activity of the plasminogen activator released by sympathetic neurons can be regulated by a normal target tissue and that this regulation may result in increased neurite outgrowth from the neurons

Vaccination with DNA plasmids expressing Gn coupled to C3d or alphavirus replicons expressing Gn protects mice against rift valley fever virus

Background: Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent. Methodology/Principal Findings: We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12. Conclusion/Significance: These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use. © 2010 Bhardwaj et al