The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of "self" versus "other" considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity.

The tumor suppressor protein p53 is known to induce either apoptosis or growth arrest depending on cellular background. We have previously reported that a bacterial redox protein azurin induces apoptosis in J774 cell line-derived macrophages whereas a site-directed mutant M44KM64E azurin shows very little cytotoxicity and fails to induce apoptosis in J774 cells. We now report that purified M44KM64E mutant azurin protein can enter both J774 cells as well as the human breast cancer MCF-7 cells. Entry of M44KM64E mutant azurin in J774 cells causes strong inhibition of cell-cycle progression at the G1 to S phase and a higher level of transcription of the p21 gene. Corresponding to high p21 levels, the levels of cyclins and cyclin-dependent kinases were greatly lowered in M44KM64E mutant azurin-treated J774 cells. Interestingly, M44KM64E mutant azurin protein failed to elicit inhibition of cell-cycle progression in MCF-7 cells, presumably because of mutation at the retinoblastoma tumor suppressor protein that allows functional E2F formation in MCF-7 cells even in the presence of high intracellular p21 level. Thus, the WT azurin induces apoptosis but little inhibition of cell-cycle progression whereas the M44KM64E mutant azurin is deficient in the induction of apoptosis but mediates strong inhibition of cell-cycle progression, demonstrating the role of a single bacterial protein and its hydrophobic patch in modulating two important functions of p53.

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Myocardial physiology in the aftermath of myocardial infarction (MI) before remodeling is an under-explored area of investigation. Here, we describe the effects of MI on the cardiac sarcomere with focus on the possible contributions of reactive oxygen species (ROS). We surgically induced MI in 6–7 month old female CD1 mice by ligation of the left anterior descending coronary artery. Data were collected 3–4 days after MI or sham surgery (SH). MI hearts demonstrated ventricular dilatation and systolic dysfunction upon echo cardiographic analysis. Sub-maximum Ca-activated tension in detergent extracted fiber bundles from papillary muscles increased significantly in the preparations from MI hearts. Ca++ sensitivity increased after MI, whereas cooperativity of activation decreased. To assess myosin enzymatic integrity we measured splitting of CaATP in myofibrillar preparations, which demonstrated a decline in CaATPase activity of myofilament myosin. Biochemical analysis demonstrated post-translational modification of sarcomeric proteins. Phosphorylation of cardiac troponin I (cTnI) and myosin light chain 2 was reduced after MI in papillary samples, as measured using a phospho-specific stain. Tropomyosin was oxidized after MI, forming disulfide products detectable by diagonal nonreducing-reducing SDS-PAGE. Our analysis of myocardial protein oxidation post-MI also demonstrated increased S-glutathionylation. We functionally linked protein oxidation with sarcomere function by treating skinned fibers with the sulfhydryl reducing agent dithiothreitol, which reduced Ca++ sensitivity in MI, but not SH, samples. Our data indicate important structural and functional alterations to the cardiac sarcomere after MI, and the contribution of protein oxidation to this process.