Cytoplasmic Irradiation Induces Mitochondrial-Dependent 53BP1 Protein Relocalization in Irradiated and Bystander Cells

The accepted paradigm for radiation effects is that direct DNA damage via energy deposition is required to trigger the downstream biological consequences. The radiation-induced bystander effect is the ability of directly irradiated cells to interact with their nonirradiated neighbors, which can then show responses similar to those of the targeted cells. p53 binding protein 1 (53BP1) forms foci at DNA double-strand break sites and is an important sensor of DNA damage. This study used an ionizing radiation microbeam approach that allowed us to irradiate specifically the nucleus or cytoplasm of a cell and quantify response in irradiated and bystander cells by studying ionizing radiation-induced foci (IRIF) formation of 53BP1 protein. Our results show that targeting only the cytoplasm of a cell is capable of eliciting 53BP1 foci in both hit and bystander cells, independently of the dose or the number of cells targeted. Therefore, direct DNA damage is not required to trigger 53BP1 IRIF. The use of common reactive oxygen species and reactive nitrogen species (RNS) inhibitors prevent the formation of 53BP1 foci in hit and bystander cells. Treatment with filipin to disrupt membrane-dependent signaling does not prevent the cytoplasmic irradiation-induced 53BP1 foci in the irradiated cells, but it does prevent signaling to bystander cells. Active mitochondrial function is required for these responses because pseudo-rho(0) cells, which lack mitochondrial DNA, could not produce a bystander signal, although they could respond to a signal from normal rho(+) cells.

Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3-22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed >= 2.3% sequence divergence between taxa and 200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.

Kir2.2 inward rectifier potassium channels are inhibited by an endogenous factor in Xenopus oocytes independently from the action of a mitochondrial uncoupler.

We previously showed inhibition of Kir2 inward rectifier K+ channels expressed in Xenopus oocytes by the mitochondrial agents carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and sodium azide. Mutagenesis studies suggested that FCCP may act via phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. This mechanism could be reversible in intact cells but not in excised membrane patches which preclude PIP2 regeneration. This prediction was tested by investigating the reversibility of the inhibition of Kir2.2 by FCCP in intact cells and excised patches. We also investigated the effect of FCCP on Kir2.2 expressed in human embryonic kidney (HEK) cells. Kir2.2 current, expressed in Xenopus oocytes, increased in inside-out patches from FCCP-treated and untreated oocytes. The fraction of total current that increased was 0.79?±?0.05 in control and 0.89?±?0.03 in 10 µM FCCP-treated (P?>?.05). Following “run-up,” Kir2.2 current was re-inhibited by “cramming” inside-out patches into oocytes. Therefore, run-up reflected not reversal of inhibition by FCCP, but washout of an endogenous inhibitor. Kir2.2 current recovered in intact oocytes within 26.5 h of FCCP removal. Injection of oocytes with 0.1 U apyrase completely depleted ATP (P?<?.001) but did not inhibit Kir2.2 and inhibited Kir2.1 by 35% (P?<?.05). FCCP only partially reduced [ATP] (P?<?.001), despite inhibiting Kir2.2 by 75% (P?<?.01) but not Kir2.1. FCCP inhibited Kir2.2 expressed in HEK cells. The recovery of Kir2.2 from inhibition by FCCP requires intracellular components, but direct depletion of ATP does not reproduce the differential inhibitory effect of FCCP. Inhibition of Kir2.2 by FCCP is not unique to Xenopus oocytes. J. Cell. Physiol. 219: 8–13, 2009. © 2008 Wiley-Liss, Inc.

Patterns of variation at a mitochondrial sequence-tagged-site locus provides new insights into the postglacial history of European Pinus sylvestris populations

Due to their maternal mode of inheritance, mitochondrial markers can be regarded as almost 'ideal' tools in evolutionary studies of conifer populations. In the present study, polymorphism was analysed at one mitochondrial intron (nad 1, exon B/C) in 23 native European Pinus sylvestris populations. In a preliminary screening for variation using a polymerase chain reaction-restriction fragment length polymorphism approach, two length variants were identified. By fully sequencing the 2.5 kb region, the observed length polymorphism was found to result from the insertion of a 31 bp sequence, with no other mutations observed within the intron. A set of primers was designed flanking the observed mutation, which identified a novel sequence-tagged-site mitochondrial marker for P. sylvestris. Analysis of 747 trees from the 23 populations using these primers revealed the occurrence of two distinct haplotypes in Europe. Within the Iberian Peninsula, the two haplotypes exhibited extensive population differentiation (Phi(ST) = 0.59; P less than or equal to 0.001) and a marked geographical structuring. In the populations of central and northern Europe, one haplotype largely predominated, with the second being found in only one individual of one population.

Lack of Oxygen Deactivates Mitochondrial Complex I IMPLICATIONS FOR ISCHEMIC INJURY?

For S-nitrosothiols and peroxynitrite to interfere with the activity of mitochondrial complex I, prior transition of the enzyme from its active (A) to its deactive, dormant (D) state is necessary. We now demonstrate accumulation of the D-form of complex I in human epithelial kidney cells after prolonged hypoxia. Upon reoxygenation after hypoxia there was an initial delay in the return of the respiration rate to normal. This was due to the accumulation of the D-form and its slow, substrate-dependent reconversion to the A-form. Reconversion to the A-form could be prevented by prolonged incubation with endogenously generated NO. We propose that the hypoxic transition from the A-form to the D-form of complex I may be protective, because it would act to reduce the electron burst and the formation of free radicals during reoxygenation. However, this may become an early pathophysiological event when NO-dependent formation of S-nitrosothiols or peroxynitrite structurally modifies complex I in its D-form and impedes its return to the active state. These observations provide a mechanism to account for the severe cell injury that follows hypoxia and reoxygenation when accompanied by NO generation.