Structural and functional characteristics of a dominant-negative isoform of porcine MHC class II transactivator.

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)-gamma-inducible isoform of porcine CIITA (pCIITA') encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA' lacks the entire GTP-binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA' this region is replaced by a 14-aa motif with homology to several signalling peptide sequences. Expression of pCIITA' in porcine (ST-IOWA) and human (HeLa) cell lines resulted in suppression of IFN-gamma-stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN-gamma-inducible variants of hCIITA, hCIITAlo and hCIITA' from Hela cells, both exhibiting dominant-negative suppression of MHC class II gene expression. Interestingly, hCIITA' encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA' including the 14-aa GTP-binding domain-replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA' and hCIITAlo sequences in Hela cells suppressed IFN-gamma-induced MHC class II gene expression. hCIITAlo, a predicted 303-aa protein with deleted GTP-binding and carboxy-terminal domain, displayed a more subtle suppression of IFN-gamma-induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full-length CIITA-mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T-cell-mediated cellular rejection.

Effect of cell surface concentration of human DAF on transgenic pig aortic endothelial cells on the degree of protection afforded against human complement deposition.

Cultures of hDAF transgenic porcine aortic endothelial cells (TPAEs) with levels of cell surface hDAF expression between 2000 and 300 000 molecules per cell have been used to determine the relationship between expression of hDAF and protection from human complement deposition in an in vitro model. At concentrations below 45 000 molecules per cell, the relationship between hDAF expression and degree of protection conferred is linear. Concentrations of 123 000 molecules per cell and higher give maximal protection (60% reduction of susceptibility to neat human serum) in this model. It is concluded that increasing hDAF expression above that displayed by the A74 line of hDAF transgenic pigs (240 000 +/- 15 000 molecules per cell) would not confer any additional benefit.

Oxytocin receptor binding activity in cultured ovine endometrium.

Oxytocin receptor binding activity in explants of caruncular and intercaruncular endometrium collected from luteal phase ewes increased during culture. An initial rise in binding activity occurred during the first 24 h of culture in both tissues; binding activity in intercaruncular endometrium continued to increase until day 6, remained unchanged on day 8 and had decreased by day 10 of culture. The maximum concentration of receptors in caruncular endometrium was significantly lower than that in intercaruncular endometrium (P < 0.001) and did not change significantly between days 4 and 10 of culture. Apparent dissociation constants and maximal binding of oxytocin receptor in cultured caruncular and intercaruncular endometrium were 3.09 and 2.72 nmol l-1 and 249 and 459 fmol [3H]oxytocin bound mg-1 protein, respectively. Concentrations of oxytocin receptor remained constant in myometrium during 96 h of culture. The rise in endometrial oxytocin receptor concentration did not result from exposure to fetal calf serum, phenol red or insulin in the culture medium. Substituting fetal calf serum with sheep serum or BSA did not block the rise in receptor binding activity. Actinomycin D and cycloheximide inhibited the rise in receptor concentration in both tissues. Co-culture of lung or kidney with endometrium had no effect on binding activity, whereas co-culture with luteal tissue effectively reduced the rise in oxytocin receptor concentration. To establish whether synthesis of functional oxytocin receptors occurred during culture, the effect of oxytocin on prostaglandin F2 alpha (PGF2 alpha) production was assessed in fresh tissue and after 48 h in culture.(ABSTRACT TRUNCATED AT 250 WORDS)