Among adults undergoing noncardiac surgery, MINS is common and associated with substantial mortality.

Despite the nontemplating nature of the abasic site, dAMP is often preferentially inserted opposite the lesion, a phenomenon commonly referred to as the "A-rule". We have evaluated the molecular mechanism accounting for this unique behavior using a thorough kinetic approach to evaluate polymerization efficiency during translesion DNA replication. Using the bacteriophage T4 DNA polymerase, we have measured the insertion of a series of modified nucleotides and have demonstrated that increasing the size of the nucleobase does not correlate with increased insertion efficiency opposite an abasic site. One analogue, 5-nitroindolyl-2'-deoxyriboside triphosphate, was unique as it was inserted opposite the lesion with approximately 1000-fold greater efficiency compared to that for dAMP insertion. Pre-steady-state kinetic measurements yield a kpol value of 126 s(-1) and a Kd value of 18 microM for the insertion of 5-nitroindolyl-2'-deoxyriboside triphosphate opposite the abasic site. These values rival those associated with the enzymatic formation of a natural Watson-Crick base pair. These results not only reiterate that hydrogen bonding is not necessary for nucleotide insertion but also indicate that the base-stacking and/or desolvation capabilities of the incoming nucleobase may indeed play the predominant role in generating efficient DNA polymerization. A model accounting for the increase in catalytic efficiency of this unique nucleobase is provided and invokes pi-pi stacking interactions of the aromatic moiety of the incoming nucleobase with aromatic amino acids present in the polymerase's active site. Finally, differences in the rate of 5-nitroindolyl-2'-deoxyriboside triphosphate insertion opposite an abasic site are measured between the bacteriophage T4 DNA polymerase and the Klenow fragment. These kinetic differences are interpreted with regard to the differences in various structural components between the two enzymes and are consistent with the proposed model for DNA polymerization.

Bcl-2 protein family members function either to promote or inhibit programmed cell death. Bcl-2, typically an inhibitor of apoptosis, has also been demonstrated to have pro-apoptotic activity (Cheng, E. H., Kirsch, D. G., Clem, R. J., et al. (1997) Science 278, 1966 -1968). The pro-apoptotic activity has been attributed to the cleavage of Bcl-2 by caspase-3, which converts Bcl-2 to a proapoptotic molecule. Bcl-2 is a membrane protein that is localized in the endoplasmic reticulum (ER) membrane, the outer mitochondrial membrane, and the nuclear envelope. Here, we demonstrate that transient expression of Bcl-2 at levels comparable to those found in stably transfected cells induces apoptosis in human embryonic kidney 293 cells and in the human breast cell line MDA-MB-468 cells. Furthermore, we have targeted Bcl-2 specifically to either the ER or the outer mitochondrial membrane to test whether induction of apoptosis by Bcl-2 is dependent upon its localization within either of these membranes. Our findings indicate that Bcl-2 specifically targeted to the mitochondria induces cell death, whereas Bcl-2 that is targeted to the ER does not. The expression of Bcl-2 does result in its cleavage to a 20-kDa protein; however, mutation of the caspase-3 cleavage site (D34A) does not inhibit its ability to induce cell death. Additionally, we find that transiently expressed ER-targeted Bcl-2 inhibits cell death induced by Bax overexpression. In conclusion, the ability of Bcl-2 to promote apoptosis is associated with its localization at the mitochondria. Furthermore, the ability of ER-targeted Bcl-2 to protect against Bax-induced apoptosis suggests that the ER localization of Bcl-2 may play an important role in its protective function.

Bcl-2 family members have been shown to be key mediators of apoptosis as either pro-or anti-apoptotic factors. It is thought that both classes of Bcl-2 family members act at the level of the mitochondria to regulate apoptosis, although the founding anti-apoptotic family member, Bcl-2 is localized to the endoplasmic reticulum (ER), mitochondrial, and nuclear membranes. In order to better understand the effect of Bcl-2 localization on its activity, we have utilized a Bcl-2 mutant that localizes only to the ER membrane, designated Bcl-2Cb5. Bcl-2Cb5 was expressed in MDA-MB-468 cells, which protected against apoptosis induced by the kinase inhibitor, staurosporine. Data presented here show that Bcl-2Cb5 inhibits this process by blocking Bax activation and cytochrome c release. Furthermore, we show that Bcl-2Cb5 can inhibit the activation of a constitutively mitochondrial mutant of Bax, indicating that an intermediate between Bcl-2 on the ER and Bax on the mitochondria must exist. We demonstrate that this intermediate is likely a BH3-only subfamily member. Data presented here show that Bcl-2Cb5 can sequester a constitutively active form of Bad (Bad3A) from the mitochondria and prevent it from activating Bax. These data suggest that Bcl-2 indirectly protects mitochondrial membranes from Bax, via BH3-only proteins.