Previous studies have demonstrated that the Dictyostelium Ga subunit Ga2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that ga2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of Ga2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and ga2 null cells carrying a series of mutant Ga2 subunits expressed from the cloned Ga2 promoter. Our results show that wild-type cells expressing Ga2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues [206][207][208][209][210] domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of downstream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant Ga2 subunit can complement ga2 null mutants. Expression of Ga2(G43V) and Ga2(G207V) have little or no effect on the effector pathways and can partially complement ga2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of Ga2(G40V) and Ga2(Q208L) are due to a constitutive adaptation of the effectors through a Ga2-mediated pathway. Analysis of PI-PLC in ga2 null mutants and in cell lines expressing mutant Ga2 proteins also strongly suggests that Ga2 is the Ga subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type Ga2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that Ga2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring Ga2 function in vivo, does not appear to be directly carried out by the Ga2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another Ga subunit or by 3y subunits released on activation of the G protein containing Ga2.

It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.

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Radiotherapy for malignant pelvic disease is often followed by acute radiation colitis (ARC). It has been reported that sucralfate treatment has a protective effect against ARC, though the mechanisms of action are unknown. The effects of sucralfate on X-ray radiation-induced apoptosis was studied at 4 Gy in the colonic crypt cells of rats. Sucralfate enemas given prior to radiation resulted in the following: (1) reduction in number of apoptotic colonic crypt cells; (2) reduction in number of caspase-3 positive cells; (3) decreases in p53 accumulation and p21 expression; (4) decreases of Bax/Bcl-2 ratio. The protective effects of sucralfate against ARC may be partially due to the suppression of radiation-induced apoptosis by way of p53 in the colon and the protection of the colonic epithelial stem cell region.

DNA-PK was inactivated by heat treatment at 44 degrees C. Ku70/Ku80, but not Ku70 alone, could restore heat-inactivated DNA-PK.