ABSTRACT
BACKGROUND AND PURPOSE: Dynamin-related protein 1 (Drp1) mediates mitochondrial fission and is thought to promote Bax/Bak-induced cytochrome c release during apoptosis. Conformationally active Bax, Bak and Bax/Bak-activating BH3-only proteins, such as Bim, are restrained by anti-apoptotic Bcl-2 proteins in cells that are 'primed for death'. Inhibition of Bcl-2/Bcl-xL/Bcl-w by the antagonist ABT-737 causes rapid apoptosis of primed cells. Hence, we determined whether Drp1 is required for cytochrome c release, respiratory alterations and apoptosis of cells that are already primed for death. EXPERIMENTAL APPROACH: We tested the Drp1 inhibitor mdivi-1 for inhibition of cytochrome c release in MCF10A cells primed by Bcl-2 overexpression. We measured ATP synthesis-dependent, -independent and cytochrome c-limited maximal oxygen consumption rates (OCRs) and cell death of immortalized wild-type (WT) and Drp1 knockout (KO) mouse embryonic fibroblasts (MEFs) treated with ABT-737. KEY RESULTS: Mdivi-1 failed to attenuate ABT-737-induced cytochrome c release. ABT-737 decreased maximal OCR measured in the presence of uncoupler in both WT and Drp1 KO MEF, consistent with respiratory impairment due to release of cytochrome c. However, Drp1 KO MEF were slightly less sensitive to this ABT-737-induced respiratory inhibition compared with WT, and were resistant to an initial ABT-737-induced increase in ATP synthesis-independent O2 consumption. Nevertheless, caspase-dependent cell death was not reduced. Pro-apoptotic Bax was unaltered, whereas Bak was up-regulated in Drp1 KO MEF. CONCLUSIONS AND IMPLICATIONS: The findings indicate that once fibroblast cells are primed for death, Drp1 is not required for apoptosis. However, Drp1 may contribute to ABT-737-induced respiratory changes and the kinetics of cytochrome c release.
Subject(s)
Cell Death/physiology , Cytochromes c/metabolism , Dynamins/physiology , Fibroblasts/metabolism , GTP Phosphohydrolases/physiology , Microtubule-Associated Proteins/physiology , Mitochondrial Proteins/physiology , Oxygen Consumption/drug effects , Proto-Oncogene Proteins c-bcl-2/physiology , Quinazolinones/pharmacology , Animals , Biphenyl Compounds/pharmacology , Cell Death/drug effects , Cells, Cultured , Cytochromes c/antagonists & inhibitors , Dynamins/antagonists & inhibitors , Dynamins/genetics , Fibroblasts/drug effects , GTP Phosphohydrolases/antagonists & inhibitors , GTP Phosphohydrolases/genetics , Humans , Mice , Mice, Knockout , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/genetics , Nitrophenols/pharmacology , Piperazines/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Sulfonamides/pharmacology , Up-Regulation , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.
Subject(s)
Chromosomes, Human, Pair 11/genetics , Radiation Hybrid Mapping/veterinary , Swine/genetics , Animals , Chromosome Mapping , Chromosomes, Artificial, Bacterial/genetics , Expressed Sequence Tags , Humans , Lod Score , Microsatellite Repeats , Radiation Hybrid Mapping/methods , Species SpecificityABSTRACT
A model is presented that enables the detailed effects of spectral broadening to be calculated for a continuous wave (CW) Doppler system by using geometric boundary arguments. The model assumes a uniform distribution of isotropic scatterers and treats the transmitter and receiver crystals as incremental sources and receivers. Detailed results for rectangular and circular geometries are presented in order to provide a physical understanding of the manner in which spectral broadening arises. Results are given for the circular geometry, to illustrate the manner in which the received spectrum is affected by the transducer size and distance from the vessel.
