Summary:We measured the temporal profile and ana tomic distribution of cells exhibiting DNA fragmentation at various durations of reperfusion after middle cerebral artery (MCA) occlusion in the rat. Focal cerebral isch emia was induced in male Wi star rats (n = 62) using an intraluminal monofilament blockade of the MCA. After 2 h of MCA occlusion, the animals were killed at different durations of reperfusion (0.5, 3, 6, 9, and 12 hand 1,2,4, 7, 14, 21, and 28 days, n = 4 per time point). Sham operated rats (n = 4) and normal rats not subjected to any surgical procedure (n = 4) were used as controls. Coronal brain sections (5 !-lm) were analyzed, using an in situ Ap opTag kit, hematoxylin and eosin, and immunohisto chemical double-staining methods. Six rats subjected to 2 h of MCA occlusion were killed at 24 h for measurement of DNA fragmentation by gel electrophoresis. Our data indicate that within a coronal section, DNA fragmenta tion was present in zero to three cells in each hemisphere of normal and sham-operated rats as well as in the conIn 1972, Kerr et aI. described a distinct type of cell death, apoptosis, that differs from necrosis in its morphological features. In contrast to necrotic cells, apoptotic cells exhibit compaction of chroma tin against the nuclear membrane, cell shrinkage with preservation of organelles, detachment from surrounding cells, and nuclear and cytoplasmic budding to form membrane-bound fragments, known as "apoptotic bodies," which are rapidly phagocytosed by adjacent parenchymal cells or

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To delineate the DCC-induced apoptotic pathway, we have identified a protein, DIP13␣, which interacts with DCC. The DIP13␣ protein has a pleckstrin homology domain and a phosphotyrosine binding domain. It interacts with a region on the DCC cytoplasmic domain that is required for the induction of apoptosis. Although ectopic expression of DIP13␣ alone causes only a slight increase in apoptosis, co-expression of DCC and DIP13␣ results in an ϳ5-fold increase in apoptosis. Removal of the DCC-interacting domain on DIP13␣ abolishes its ability to enhance DCC-induced apoptosis. Inhibition of endogenous DIP13␣ expression by small interfering RNA blocks DCC-induced apoptosis. Our data suggest that DIP13␣ is a mediator of the DCC apoptotic pathway.The candidate tumor-suppressor gene deleted in colorectal cancer (DCC) 1 was first cloned from a locus on chromosome arm 18q where allelic deletions occur in over 70% of primary colorectal tumors (1). Since that time, loss of heterozygosity at the DCC locus and loss of DCC expression have been shown in many other tumor types (2) including prostate carcinomas (3). The loss of DCC expression has therefore been associated with tumor progression. Although the known tumor suppressor gene MADH4/DPC4/Smad4 is mapped in close proximity to the DCC locus (4, 5), re-evaluation of loss of heterozygosity at 18q in colon tumor samples indicated that DCC, but not Smad4, was the most frequently altered gene on chromosome 18q13.3-21.3 (6). In a study of 115 pancreatic and 14 biliary cancers for homozygous deletions of DCC exons and flanking 18q regions, seven homozygous deletions were seen in the region that includes the DCC gene. In fact, DCC was the only known gene affected by all seven deletions. In two tumors, the deletions inactivate DCC but not Smad4 (7). These loss of heterozygosity and mutational data support the hypothesis that DCC acts as a tumor suppressor.DCC encodes a type I membrane protein that falls into a subgroup of the immunoglobulin superfamily (8). We and others have shown that DCC may exert its tumor-suppression function through induction of apoptosis (9, 10). DCC and its orthologs, UNC-40 in Caenorhabditis elegans and frazzled in Drosophila, have been established as receptors for netrin-1 and play an important role in axon outgrowth and cell migration in the developing nervous system (11)(12)(13)(14). It has been shown that induction of apoptosis by DCC can be blocked by netrin-1 (10). Expression of DCC colocalizes with areas of apoptotic precerebellar neurons in netrin-1 Ϫ/Ϫ mice, whereas apoptosis is absent in the same DCC-expressing areas in wild-type mice (15). Therefore DCC may induce cell death in settings where ligand is unavailable. Consistent with this view, netrin-1 knockout mice grow fewer cells particularly in the developing brainstem (16, 17).Very little is known about the apoptotic signaling of DCC. Unlike the other well characterized receptors such as Fas and tumor necrosis factor receptor, no apparent death domain can be identified in the DCC cytoplasmic r...

Preterm birth (PTB) is a leading cause of neonatal death worldwide1. Intrauterine and systemic infection and inflammation cause 30–40% of spontaneous preterm labor (PTL)2, which precedes PTB. Although antibody production is a major immune defense mechanism against infection, and B cell dysfunction has been implicated in pregnancy complications associated with PTL3,4, the functions of B cells in pregnancy are not well known5–8. We found that choriodecidua of women undergoing spontaneous PTL harbored functionally altered B cell populations. B cell–deficient mice were markedly more susceptible than wild-type (WT) mice to PTL after inflammation, but B cells conferred interleukin (IL)-10-independent protection against PTL. B cell deficiency in mice resulted in a lower uterine level of active progesterone-induced blocking factor 1 (PIBF1), and therapeutic administration of PIBF1 mitigated PTL and uterine inflammation in B cell–deficient mice. B cells are a significant producer of PIBF1 in human choriodecidua and mouse uterus in late gestation. PIBF1 expression by B cells is induced by the mucosal alarmin IL-33 (ref. 9). Human PTL was associated with diminished expression of the a-chain of IL-33 receptor on choriodecidual B cells and a lower level of active PIBF1 in late gestation choriodecidua. These results define a vital regulatory cascade involving IL-33, decidual B cells and PIBF1 in safeguarding term pregnancy and suggest new therapeutic approaches based on IL-33 and PIBF1 to prevent human PTL.

Thioredoxin-interacting protein (TXNIP) plays a critical role in oxidative stress, inflammation, apoptosis and the pathogenesis of diabetic retinopathy (DR). However, the role of TXNIP in high glucose-induced retinal pigment epithelium (RPE) dysfunction is still unknown. Here, we show that high glucose (HG; 25 mM,) significantly increases TXNIP expression at both the mRNA and protein levels when compared to low glucose (LG; 5.5 mM) in a human RPE cell line (ARPE-19) and primary human RPE (HRPE) cells. TXNIP upregulation is associated with mitochondrial membrane depolarization, fragmentation and mitophagic flux to lysosomes. We used confocal live-cell imaging of RPE cells expressing mt-Keima, a coral protein that emits green light in mitochondria (alkaline or neutral pH) and red light in the acidic lysosome, to measure mitophagic flux. We observed an elongated mitochondrial network of green mt-Keima under LG, which is fragmented in HG. Red mt-Keima accumulates in lysosomes as small punctate aggregations under LG in both ARPE-19 and HRPE cells, whereas they are significantly enlarged (two- to threefold) under HG. Lysosomal enlargement under HG is further illustrated by lysosomal membrane protein LAMP1-mCherry expression in both ARPE-19 and HRPE cells. Furthermore, HG causes lysosomal cathepsin L inactivation and pro-inflammatory caspase-1 activation in ARPE-19 cells. TXNIP knockdown by shRNA prevents mitochondrial fragmentation, mitophagic flux and lysosome enlargement under HG. In addition, antioxidant N-acetylcysteine (NAC) and Amlexanox (Amlx), an inhibitor of protein kinase TBK1 and of the mitophagic adaptors Optineurin (Optn) and Sequestosome 1 (p62/SQSTM1), prevent mitophagic flux and lysosome enlargement. These results suggest that TXNIP mediates several deleterious effects of high glucose on RPE, which may be implicated in the development of DR.