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DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide-induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs. [Cancer Res 2009;69(21):8356-65]

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca 2؉-activated Cl ؊ current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl ؊ current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G␤␥-binding proteins. In addition, we examined which of mammalian PLC␤1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl ؊ current. Injection of G␣ q or G␣ 11 cRNA increased the basal Cl ؊ current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl ؊ current, whereas G␣ i2 and G␣ oA cRNA injection had no significant effect. The changes following G␣ q cRNA injection were prevented when G␤ 1 ␥ 2 and G␣ q subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G␣ q Q209L, a constitutively active mutant that does not bind to G␤␥, produced effects similar to those of G␣ q cRNA injection. The effects of G␣ q Q209L cRNA injection, however, were not prevented by co-injection of G␤ 1 ␥ 2 cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G␣ q/11 among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G␣ subunit, resulted in a severe attenuation of ginsenoside effect on the Cl ؊ current. Finally, antibodies against PLC␤3, but not -␤1 and -␤2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G␣ q/11 coupled to mammalian PLC ␤3-like enzyme mediates ginsenoside effect on Ca 2؉

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Pooled genomic DNA from 10 dogs was subjected to polymerase chain reaction with primers targeting the retroviral pro/pol region. Sequence analysis of 120 clones obtained by PCR revealed 81 of retroviral origin. Subsequent analysis of the dog genome (CanFam 2.0) by BLAST investigation using degenerate PCR products and previously identified retroviral sequences permitted the identification of additional retroviral γ and β sequences. A phylogenetic analysis using the retroviral protease (PR) and reverse transcriptase (RT) sequences in the dog genome resulted in identification of 17 γ and 7 β families. In addition, we also identified 167 spuma-like ERV elements from CanFam 2.0 based on sequence homology to murine (Mu)ERV-L and human (H)ERV-L. Our results could contribute to the understanding of the influence of retroviruses in shaping the genome structure and altering gene expression by providing quantitative and locational information of ERV loci and their diversity in the dog genome.

In an attempt to enable comprehensive high-resolution genotyping of the swine leukocyte antigen (SLA) gene, we performed a systemic analysis of nucleotide polymorphisms at introns 1 and 2 and exon 2 from diverse alleles of SLA-DRB1 and DRB1 pseudogenes. We amplified and cloned 16 partial sequences of SLA-DRB1 and DRB2 introns 1 and 2 from different alleles, and analyzed them together with sequences of four reported SLA-DRB pseudogenes, DRB2, 3, 4, and 5. The results showed the presence of extreme nucleotide variations within introns 1 and 2 of SLA-DRB-related genes including substitutions and deletions. On the basis of these results, we developed a comprehensive genotyping method for SLA-DRB1 by genomic polymerase chain reaction (PCR) and subsequent direct sequencing. A total of 415 animals were genotyped and 67 allelic combinations from 18 DRB1 alleles were identified. Among them, two alleles, SLA-DRB1*kn04 and *kn05, were previously unreported. SLA-DRB1 genotyping results from this study combined with those of SLA-DQB1 from our previous study presented 10 SLA class II haplotypes, three of which were previously unreported. Population analysis using seven different pig breeds showed differences in the allele frequency of SLA-DRB1 among breeds. Our results should benefit biological experiments requiring sequence-level genotyping results of SLA-DRB1 and further study of the complete genetic diversity of SLA-DRB1 using field samples.

lator of G protein signaling (RGS) family members, such as RGS2, interact with G␣ subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and attenuating the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. They are also reported to regulate G protein-effector interactions and form multiprotein signaling complexes. Ischemic stress-induced changes in RGS2 expression have been described in astrocytes, and these changes are associated with intracellular signaling cascades, suggesting that RGS2 upregulation may be an important mechanism by which astrocytes may regulate RGS2 function in response to physiological stress. However, information on the functional roles of stress-induced modulation of RGS2 protein expression in astrocyte function is limited. We report the role of ischemic stress in RGS2 protein expression in rat C6 astrocytoma cells and primary mouse astrocytes. A marked increase in RGS2 occurred after ischemic stress induced by chemicals (sodium azide and 2-deoxyglucose) or oxygen-glucose deprivation (OGD, real ischemia). RGS2 mRNA expression was markedly enhanced by 1 h of exposure to chemical ischemia or 6 h of OGD followed by 2 or 6 h of recovery, respectively. This enhanced expression in primary astrocytes and C6 cells was restored to baseline levels after 12 h of recovery from chemically induced ischemic stress or 4 -6 h of recovery from OGD. RGS2 protein was also significantly expressed at 12-24 h of recovery from ischemic insult. Ischemiainduced RGS2 upregulation was associated with enhanced apoptosis. It significantly increased annexin V-positive cells, cleaved caspase-3, and enhanced DNA ladder formation and cell cycle arrest. However, a small interfering RNA (siRNA)-mediated RGS2 knockdown reversed the apoptotic cell death associated with ischemia-induced RGS2 upregulation. Upregulated RGS2 was significantly inhibited by SB-203580, a p38 MAPK inhibitor. Rottlerin, a potent inhibitor of PKC␦, completely abrogated the increased RGS2 expression. We also examine whether ischemia-induced RGS2-mediated apoptosis is affected by siRNA-targeted endogenous PKC␦ downregulation or its phosphorylation. Although RGS2 upregulation was not affected, siRNA transfection significantly suppressed endogenous PKC␦ mRNA and protein expressions. Ischemia-induced PKC␦ phosphorylation and caspase-3 cleavage were dose dependently inhibited by PKC␦ knockdown, and this endogenous PKC␦ suppression reversed ischemia-induced annexin V-positive cells. This study suggests that ischemic stress increases RGS2 expression and that this condition contributes to enhanced apoptosis in C6 cells and primary astrocytes. The signaling it follows may involve PKC␦ and p38 MAPK pathways. stress; protein kinase C; p38 MAPK; gliocytoma ASTROCYTES, THE MOST ABUNDANT glial cell type in the brain, provide metabolic and trophic support to neurons and modulate synaptic activity (52). It has been reported that astrocytes play an important role in the metabolism of neurotransmitters and in ...