Biological activity and in vivo half-life of pro-activin A in male rats.

Mature TGF-ß proteins are used in vivo to promote bone growth, combat obesity, reverse fibrosis and pulmonary arterial hypertension, and as potential rejuvenation factors. However, the serum half-life of this family of growth factors is short (∼5 min), limiting their therapeutic potential. Because TGF-ß proteins are normally secreted from cells with their prodomains attached, we considered whether these molecules could extend the in vivo half-life and activity of their respective growth factors. Using activin A as a model ligand, we initially modified the cleavage site between the pro- and mature domains to ensure complete processing of the activin A precursor. Co-immunoprecipitation studies confirmed mature activin A is secreted from cells in a non-covalent complex with its prodomain, however, the affinity of this interaction is not sufficient to suppress activin A in vitro biological activity. The plasma clearance profiles of purified pro- and mature activin A were determined over a 4 h period in adult male rats. Both activin forms demonstrated a two-phase decay, with the half-life of pro-activin A (t1/2 fast = 12.5 min, slow = 31.0 min) being greater than that of mature activin A (t1/2 fast = 5.5 min, slow = 20.3 min). Both pro- and mature activin A induced significant increases in serum follicle stimulating hormone levels after 4 h, but no differences were observed in the relative in vivo bioactivities of the two activin isoforms. Increased serum half-life of activin A in the presence of its prodomain identifies a new means to increase the therapeutic effectiveness of TGF-ß proteins.

Construction of an infectious cDNA clone of Enterovirus 71: insights into the factors ensuring experimental success.

Enterovirus 71 (EV 71) is a causative agent of mild Hand Foot and Mouth Disease but is capable of causing severe complications in the CNS in young children. Reverse genetics technology is currently widely used to study the pathogenesis of the virus. The aim of this work was to determine and evaluate the factors which can contribute to infectivity of EV 71 RNA transcripts in vitro. Two strategies, overlapping RT-PCR and long distance RT-PCR, were employed to obtain the full-length genome cDNA clones of the virus. The length of the poly(A) tail and the presence of non-viral 3'-terminal sequences were studied in regard to their effects on infectivity of the in vitro RNA transcripts of EV 71 in cell culture. The data revealed that only cDNA clones obtained after long distance RT-PCR were infectious. No differences were observed in virus titres after transfection with in vitro RNA harbouring a poly(A) tail of 18 or 30 adenines in length, irrespective of the non-viral sequences at the 3'-terminus.