Detailed analytical subcellular fractionation of non-pregnant porcine corpus luteum reveals peroxisomes of normal size and significant UDP-glucuronosyltransferase activity in the high-speed supernatant

Boström, Malin; Björk, Karl; Nelson, Bruce R.; DePierre, Joseph W.

doi:10.1016/j.cbi.2003.10.002

Cited by 1 publication

(1 citation statement)

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“…Measurements of lactate dehydrogenase were performed using a kit (SigmaAldrich) according to the instruction provided by the manufacturer. Based on Bostrom et al [2004], mitochondria with lactate dehydrogenase activity less than 5% were used for present experiments.…”

Section: Preparation Of Mitochondriamentioning

confidence: 99%

Generation of reactive oxygen species is an early event in dolichyl phosphate‐induced apoptosis

Yokoyama

Nohara

Okubo

et al. 2006

J of Cellular Biochemistry

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The mechanism of induction of apoptosis by dolichyl phosphate (Dol-P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 microg/ml of Dol-P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol-P, but nearly 50% of activity still remained at 20 microg/ml. Dol-P induced release of cytochrome-c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time-dependent manner. Flow cytometric analysis also indicated that Dol-P caused loss of mitochondrial membrane potential (Deltapsi(m)) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol-P-induced ROS generation and activation of caspase-3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol-P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol-P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol-P-induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol-P-induced apoptosis.

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Section: Preparation Of Mitochondriamentioning

confidence: 99%