RNA editing sites in plant mitochondria can share cis-elements

RNA editing in flowering plant mitochondria alters numerous C nucleotides in a given mRNA molecule to U residues. To investigate whether neighbouring editing sites can influence each other we analyzed in vitro RNA editing of two sites spaced 30 nt apart. Deletion and competition experiments show that these two sites carry independent essential specificity determinants in the respective upstream 20-30 nucleotides. However, deletion of a an upstream sequence region promoting editing of the upstream site concomitantly decreases RNA editing of the second site 50-70 nucleotides downstream. This result suggests that supporting cis-/trans-interactions can be effective over larger distances and can affect more than one editing event.     

Changes in the texture and viscoelastic properties of bread containing rice porridge during storage

The objective of this study was to investigate the effects of rice porridge on the texture and viscoelastic properties of bread during storage. Three types of bread, wheat flour bread, 15% rice flour bread, and 15% rice porridge bread, were prepared. After baking and storing the bread for 24 h, 48 h, and 72 h at room temperature, we measured the texture and viscoelastic properties of the bread crumbs by texture profile analysis (TPA) and creep test. The 15% rice porridge bread showed a significantly higher specific volume and maintained softer crumbs than the other two types (p<0.05). It also had a slightly stickier texture than the others. It can be concluded that rice porridge improves the specific volume, texture, and viscoelastic properties of bread crumbs during storage.     

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2⁺ cells after intravenous injection and in CD11c⁺ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8⁺ T-cell response in HLA-A2 transgenic mice and stimulated a CD4⁺ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A^b. As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.     

Life cycle polyphenism as a factor affecting ecological divergence within <Emphasis Type="Italic">Notophthalmus viridescens</Emphasis>

Polyphenism, which allows a single genotype to express multiple discrete phenotypes in response to environmental cues, is an adaptive trait in heterogeneous environments. Pond hydroperiod is an important ecological parameter affecting amphibian life history, and variation in local pond hydrology has been hypothesized to play a role in species divergence via changes in polyphenism. The eastern newt (Notophthalmus viridescens) expresses life cycle polyphenism. Larvae develop along three possible pathways: metamorphosis to aquatic lunged adult via a terrestrial juvenile (eft) stage, metamorphosis directly to an aquatic lunged adult, or maturation directly to an aquatic gilled adult without metamorphosis (i.e., paedomorphosis). Subspecies of N. viridescens vary in their polyphenic patterns, suggesting possible adaptation to different environments. However, no studies have experimentally tested how genetic and environmental components contribute to the observed differences among subspecies and whether such differences may facilitate divergence. We tested whether adaptation to local pond hydrology via polyphenic changes existed among subspecies by rearing larvae of three subspecies (N. v. dorsalis, N. v. louisianensis, and N. v. viridescens) along three hydroperiod regimes (short, long, and constant) in outdoor artificial ponds. We found that larval N. v. viridescens obligately metamorphosed to efts under all hydroperiods, whereas N. v. dorsalis and N. v. louisianensis exhibited plasticity: larvae metamorphosed to efts under drying conditions but metamorphosed directly to aquatic adults or became paedomorphic in constant water. Also, N. v. viridescens metamorphosed to efts faster and at a smaller body size than the other two subspecies. These data suggest that subspecies of N. viridescens are adapted to different pond hydroperiods, supporting the potential for polyphenism to facilitate divergence. Canalizing selection for certain alternative phenotypes within a single species in which other populations remain plastic may play an important role in the initiation of ecological divergence.     

Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.     

Oxidation of mitochondrial deoxynucleotide pools by exposure to sodium nitroprusside induces cell death

Human MutT homolog (hMTH1) hydrolyzes oxidized purine nucleoside triphosphates to monophosphates, thereby avoiding incorporation of such oxidized purines into DNA or RNA. We examined whether hMTH1 prevents cellular dysfunction induced by sodium nitroprusside, a spontaneous NO donor. Exposure to sodium nitroprusside caused an 8-oxoguanine (8-oxoG) buildup in DNA of proliferating MTH1-null cells which underwent mitochondrial degeneration and subsequently died. Quiescent MTH1-null cells also died with 8-oxoG buildup but only when the buildup affected mitochondrial and not nuclear DNA. In both proliferative and quiescent conditions, the accumulation of 8-oxoG in DNA and cell death was effectively prevented by hMTH1. Knockdown of MUTYH in quiescent MTH1-null cells significantly prevented the cell death, suggesting that 8-oxoG incorporated into mitochondrial DNA is a main cause of this form of cell death. To verify this possibility, an artificially modified hMTH1, namely mTP-EGFP-hMTH1, which localizes exclusively in mitochondria, was expressed in MTH1-null cells. mTP-EGFP-hMTH1 selectively prevented buildup of 8-oxoG in mitochondrial but not nuclear DNA after exposure of proliferating cells to sodium nitroprusside, and also efficiently prevented cell death. We thus concluded that exposure of cells to sodium nitroprusside causes oxidation of mitochondrial deoxynucleotide pools, and that buildup of oxidized bases in mitochondrial DNA initiates cell death.     

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(−)]. At 4 and 8 weeks, VC levels in brains from VC(−) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(−) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.     

Functional cooperation among Ras, STAT5, and phosphatidylinositol 3-kinase is required for full oncogenic activities of BCR/ABL in K562 cells.

BCR/ABL tyrosine kinase generated from the chromosomal translocation t(9;22) causes chronic myelogenous leukemia and acute lymphoblastic leukemia. To examine the roles of BCR/ABL-activated individual signaling molecules and their cooperation in leukemogenesis, we inducibly expressed a dominant negative (DN) form of Ras, phosphatidylinositol 3-kinase, and STAT5 alone or in combination in p210 BCR/ABL-positive K562 cells. The inducibly expressed DN Ras (N17), STAT5 (694F), and DN phosphatidylinositol 3-kinase (Delta p85) inhibited the growth by 90, 55, and 40%, respectively. During the growth inhibition, the expression of cyclin D2 and cyclin D3 was suppressed by N17, 694F, or Delta p85; that of cyclin E by N17; and that of cyclin A by Delta p85. In addition, N17 induced apoptosis in a small proportion of K562, whereas 694F and Delta p85 were hardly effective. In contrast, coexpression of two DN mutants in any combinations induced severe apoptosis. During these cultures, the expression of Bcl-2 was suppressed by N17, 694F, or Delta p85, and that of Bcl-XL by N17. Furthermore, although K562 was resistant to interferon-alpha- and dexamethasone-induced apoptosis, disruption of one pathway by N17, 694F, or Delta p85 sensitized K562 to these reagents. These results suggested that cooperation among these molecules is required for full leukemogenic activities of BCR/ABL.