The mRNA processing body (P-body) is a cellular structure that regulates gene expression by degrading cytoplasmic mRNA. The objective of this study was to identify and characterize novel components of the mammalian P-body. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis have antibodies directed against this structure. Serum from one of these patients was used to identify a cDNA encoding Ge-1, a 1401-amino-acid protein. Ge-1 contains an N-terminal WD40 motif and C-terminal domains characterized by a repeating c(X 2-3 ) motif. Ge-1 co-localized with previously identified P-body components, including proteins involved in mRNA decapping (DCP1a and DCP2) and the autoantigen GW182. The Ge-1 C-terminal domain was necessary and sufficient to target the protein to P-bodies. Following exposure of cells to oxidative stress, Ge-1-containing P-bodies were found adjacent to TIA-containing stress granules. During the recovery period, TIA returned to the nucleus while Ge-1-containing P-bodies localized to the perinuclear region. siRNA-mediated knock-down of Ge-1 resulted in loss of P-bodies containing Ge-1, DCP1a, and DCP2. In contrast, Ge-1-containing P-bodies persisted despite knock-down of DCP2. Taken together, the results of this study show that Ge-1 is a central component of P-bodies and suggest that Ge-1 may act prior to the 5 0 -decapping step in mRNA degradation.

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The mRNA processing body (P-body) is a cellular structure that has an important role in mRNA degradation. P-bodies have also been implicated in RNAi-mediated post-transcriptional gene silencing. The objective of this study was to identify and characterize novel components of the mammalian P-body. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis have antibodies directed against this structure. Serum from one of these patients was used to identify a cDNA encoding RAP55, a 463-amino acid protein. RAP55 colocalized with previously identified P-body components DCP1a and Ge-1. RAP55 contains an N-terminal Sm-like domain and two C-terminal RGG-rich domains separated by an FDF motif. The two RGG domains and the FDF domain were necessary and sufficient to target the protein to P-bodies. A fragment of RAP55 consisting of the FDF and the second RGG domains did not localize to P-bodies, but was able to displace other P-body components from this structure. After cells were subjected to arsenite-induced stress, RAP55 was detected in TIA-containing stress granules. The second RGG domain was necessary and sufficient for stress granule localization. siRNA-mediated knock-down of RAP55 resulted in loss of P-bodies, suggesting that RAP55 acts prior to the 5¢-decapping step in mRNA degradation. The results of this study show that RAP55 is a component of P-bodies in cells at rest and localizes in stress granules in arsenite-treated cells. RAP55 may serve to shuttle mRNAs between P-bodies and stress granules.

Purpose:To determine if oxidative and nitrative stress and/or apoptosis contribute to increased coagulation when combining radiofrequency (RF) ablation with liposomal doxorubicin. Materials and Methods:Animal care committee approval was obtained. R3230 mammary adenocarcinomas in Fischer rats were treated with either RF ablation ( n = 43), 1 mg of intravenously injected liposomal doxorubicin ( n = 26), or combined therapy ( n = 30) and were compared with control subjects ( n = 11). A subset of animals receiving combination therapy ( n = 24) were treated in the presence or absence of N -acetylcysteine (NAC) administered 24 hours and 1 hour before RF ablation. Tumors were analyzed 2 minutes to 72 hours after treatment to determine the temporal range of response by using immunohistochemical staining of the apoptosis marker cleaved caspase-3, phosphorylated g H2AX , and Results:By 4 hours after RF ablation alone, a 0.48-mm 6 0.13 (standard deviation ) peripheral band with 57.0% 6 7.3 cleaved caspase-3 positive cells was noted at the ablation margin, whereas a 0.73-mm 6 0.18 band with 77.7% 6 6.3 positivity was seen for combination therapy ( P , .03 for both comparisons). Combination therapy caused increased and earlier staining for 4-HNE-modifi ed proteins, 8-OHdG, NT, and g H2AX with colocalization to cleaved caspase-3 staining. A rim of increased HSP70 was identifi ed peripheral to the area of cleaved caspase-3. Parameters of oxidative and nitrative stress were signifi cantly inhibited by NAC 1 hour following RF ablation, resulting in decreased cleaved caspase-3 positivity (0.28-mm 6 0.09 band of 25.9% 6 7.4 positivity vs 0.59-mm 6 0.11 band of 62.9% 6 6.0 positivity, P , .001 for both comparisons). Conclusion:Combining RF ablation with liposomal doxorubicin increases cell injury and apoptosis in the zone of increased coagulation by using a mechanism that involves oxidative and nitrative stress that leads to accelerated apoptosis.q RSNA, 2010

The Epstein-Barr virus (EBV) EBNA-LP protein is important for EBV-mediated B-cell immortalization and is a potent gene-specific coactivator of the viral transcriptional activator, EBNA2. The mechanism(s) by which EBNA-LP functions as a coactivator remains an important question in the biology of EBV-induced B-cell immortalization. In this study, we found that EBNA-LP interacts with the promyelocytic leukemia nuclear body (PML NB)-associated protein Sp100 and displaces Sp100 and heterochromatin protein 1a (HP1a) from PML NBs. Interaction between EBNA-LP and Sp100 was mediated through conserved region 3 in EBNA-LP and the PML NB targeting domain in Sp100. Overexpression of Sp100 lacking the N-terminal PML NB targeting domain, but not a mutant form of Sp100 lacking the HP1a interaction domain, was sufficient to coactivate EBNA2 in a gene-specific manner independent of EBNA-LP. These findings suggest that Sp100 is a major mediator of EBNA-LP coactivation. These studies indicate that modulation of PML NB-associated proteins may be important for establishment of latent viral infections, and also identify a convenient model system to investigate the functions of Sp100.