We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the Cterminal five-residue segment (C-segment) contained the information required for mitochondrialtargeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.

Positive remodeling was more frequently observed in ACS than in stable CAD. Moreover, the degree of positive remodeling was greater in AMI than in UAP. These results may reflect the impact of remodeling types and its degree in the culprit lesion of CAD on clinical presentation.

No abstract

-This review showed the common pathogenic mechanism in the development of non-alcoholic or alcoholic steatohepatitis. In particular, we describe the role of innate immune system and oxidative stress caused by gut-derived endotoxin. Gut-derived endotoxin plays an important role in alcoholic liver injury. It was reported that acute ethanol administration reduced activation of Kupffer cells. It is therefore possible that alcohol-induced hepatocellular damage occurs as a result of bacterial or endotoxin translocation under a reduction of the reticuloendothelial system (RES) function in alcoholic liver disease (ALD). On the other hand, recently, attention has been directed toward the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut-derived endotoxin via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol-induced liver injury. It is generally accepted that activation of the innate immune system and increased release of proinflammatory cytokines and other mediators plays an important role in the development of ALD. It was shown that Kupffer cells activation by endotoxin via Toll-like receptor (TLR-4) is involved in alcoholinduced liver injury and that ethanol-induced oxidative stress is important in the regulation of transcription factor NF-κB activation and that cytokine production by Kupffer cells. TNF-α and free radicals are produced in early alcohol-induced liver injury. In support of this finding, the pathology caused by alcohol was blocked nearly completely in TNF-α receptor 1. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of the CYP2E1 form of cytochrome P450 enzymes by ethanol. Initial efforts to clarify the mechanisms that promote the progression from steatosis to steatohepatitis somewhat artificially divides disease mechanisms into "first and second" hit. The best candidates for these second hits were considered to be oxidative stress (CYP2E1 induction) and associated lipid peroxidation and cyokines, principally, TNF-α. Some of the most definitive data on the importance of the innate immune system or oxidative stress in the pathogenesis of liver disease come from studies of alcoholic and non-alcoholic steatohepatitis in animals.

Apoptosis in activated T cells in vivo requires the proapoptotic Bcl-2 family member Bim. We show here that, despite its ability to bind LC8, a component of the microtubule dynein motor complex, most of the Bim in both healthy and apoptotic T cells is associated with mitochondria, not microtubules. In healthy resting T cells Bim is bound to the antiapoptotic proteins Bcl-2 and Bcl-x L. In activated T cells, levels of Bcl-2 fall, and Bim is associated more with Bcl-x L and less with Bcl-2. Our results indicate that, in T cells, Bim function is regulated by interaction with Bcl-2 family members on mitochondria rather than by sequestration to the microtubules. During immune responses, antigen-specific T cells expand in large numbers and then die (1-3). Sometimes this death involves death receptors (4). However, under some circumstances, the death of activated T cells is driven by changes in the activity of Bcl-2-related proteins.Bcl-2-related proteins are classified by the presence or absence in their sequences of BH1-4 domains. The antiapoptotic family members contain all four of these domains. Family members that are thought to be the executioners of cell death, Bax and Bak, contain only BH1-3. Many family members express only BH3 regions. These proteins are also proapoptotic.It is not clear how Bcl-2 family proteins kill activated T cells. One hypothesis suggests that apoptotic stimuli activate BH3-region-only proteins and these proteins in turn activate Bax or Bak, either by direct interaction, or by neutralizing the antiapoptotic Bcl-2-like proteins (5-7). Alternatively, it has been suggested that antiapoptotic proteins such as Bcl-2 may inhibit the activity of death-dealing caspases. In this model, proapoptotic proteins act by binding to Bcl-2, thus interfering with its inhibition of the caspases.Apoptosis of activated T cells requires the BH3-only Bcl-2 family member Bim (8); however, the processes that regulate Bim activity in T cells are unknown. Some cell types express low levels of Bim constitutively and rapidly increase its levels when they die (9, 10). However, healthy T cells contain appreciable levels of Bim that do not change very much when the cell is activated and undergoes Bim-dependent death (8). In other cell types, Bim activity is controlled by its location. Bim binds with high affinity to LC8, a component of the microtubule-associated dynein motor complex (11). Thus, in some cells, when healthy, Bim is sequestered to the microtubular dynein motor complex. In these cells, death-inducing signals cause release of Bim plus LC8 from the dynein motor complex, translocation of the complex to mitochondria, and Bim-induced death of the cell (11).In this study, we tested the idea that Bim might move from microtubules to mitochondria during activation-induced T cell death. Surprisingly, we found that Bim was not bound to microtubules, even in healthy cells, but rather was associated with mitochondria. In both healthy and dying T cells, Bim was already bound to Bcl-2 and Bcl-x L although the ratio of bin...