24 hrs later, the cells were treated with 5 [micro]g CTA1 or 5 and 10 [micro]g TCTA1T and incubated in CO2 incubator for 2 hrs.
The 96-well ELISA plates were precoated with CTA1 (1 [micro]g/ml) or CT (2 [micro]g/ml) and CTA1- or CT-specific Ab titers were determined by ELISA as described above.
Accordingly, we constructed our candidate adjuvant TCTA1T by fusing HIV-1 Tat PTD onto both N-terminus and C-terminus of CTA1 subunit, which consequently allowed bypassing of CTB-dependent cellular internalization of CTA1 (Figure 1(a)).
To investigate whether TCTA1T possesses the capacity to translocate into target cells, HeLa cells were treated with 5 or 10 [micro]g TCTA1T or 5 [micro]g wild-type CTA1 (lacking HIV-1 Tat PTD fusion) for 2hrs.
Meanwhile, the levels of OVA-specific serum IgG1 and IgG2a titers elicited by CTA1 were 4-fold lower than those induced by TCTA1T (data not shown).
CTT1, but Not CTA1, Restores the Lifespan of MET18Deficient Yeast.
Our results suggest that MET18 deficiency increases the cellular response of yeast to [H.sub.2][O.sub.2] and CHP and shortens RLS through downregulation of catalases CTT1 and CTA1.
In our study, the total catalase activity as well as the mRNA levels of both CTT1 and CTA1 was significantly decreased in MET18/met18[DELTA] mutant compared with the WT strain (Figures 2(b) and 2(c)), resulting in an increased level of intracellular [H.sub.2][O.sub.2] in mutant cells (Figure 2(d)).
Accordingly, the increased [H.sub.2][O.sub.2] level and enhanced response to oxidative stress have been observed in yeast cells lacking CTA1 and CTT1 [24, 25], which is consistent with our findings.
Some studies show that the activity of CTT1 is essential to protect yeast cells against [H.sub.2][O.sub.2] challenge, whereas the activity of CTA1 is dispensable .
The entry of CTA1 to the cell cytosol is the key step for intoxication because CTA1 catalyzes the ADP ribosylation of the trimeric Gsa component of AC.
A different approach has been taken by Lycke (50), who instead of attenuating the A-subunit made a gene fusion protein between fully active CTA1 and a Staphylococcus aureus protein-A derivative named DD (Fig.