Flow Cytometric Analysis of Ca2+-Induced Membrane Permeability Transition of Isolated Rat Liver Mitochondria

The membrane permeability transition (MPT) of mitochondria plays an important role in the mechanism of apoptotic cell death in various cells. Classic type MPT is induced by Ca2+ in the presence of inorganic phosphate and respiratory substrate, and is characterized by various events including generation of reactive oxygen species (ROS), membrane depolarization, swelling, release of Ca2+ and high sensitivity to cyclosporine A. However, the sequence of these events and the effect of antioxidants on their events remain obscure. Flow cytometry is a convenient method to investigate the order of events among various functions occurring in MPT using a limited amount of mitochondria (200 µl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca2+ sequentially induced ROS generation, depolarization, swelling and Ca2+ release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca2+-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca2+-induced phospholipase A2 suppressed all of the events associated with Ca2+-induced MPT. These results suggested that ROS generation and phospholipase A2 activation by Ca2+ underlie the mechanism of the initiation of MPT

Studies on the fluorescent dye, diS-C(3)-(5): monitoring of membrane potentials and inhibition of endogenous respiration in Ehrlich ascites tumor cells

Changes in fluorescent intensity of the cyanine dye, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide [diS-C(3)-(5)], were monitored in Ehrlich ascites tumor cells (EATC) in correlation with glycolytic energy metabolism and endogenous respiration. After addition of glucose to EATC, membrane potentials monitored by diS-C(3)-(5) increased. The cyanine dyes were incorporated into EATC in proportion to the membrane potentials, and inhibited the endogenous respiration of EATC by more than 85% at a concentration of 100 μM. The inhibited respiration was partially released by addition of an uncoupler of oxidative phosphorylation (DNP). The respiratory activity of NAD-linked substrates in mitochondria (2.7 mg protein/ml) was also inhibited by diS-C(3)-(5), which inhibited the Rotenone-sensitive sites of the respiratory chains (Site 1) and increased Mg(++)ATPase activity. The inhibition by diS-C(3)-(5) of endogenous respiration in EATC in vivo was also effective even 24 hours after administration of the dye into the peritoneal cavity. The mean survival times of EATC-bearing mice were significantly prolonged by the administration of diS-C(3)-(5). The data suggest that the cell membrane potential is a useful factor when administering therapeutic drugs into target cells

Diagnostic evaluation for gynecologic tumor with ultrasonic tomography. - with special reference to ovarian tumor

Diagnostic value for ovarian tumor with Contact Compound Scope of the ultrasonic tomography is discussed. The size and localization of the tumor are relatively easy to determin with ultrasonic equipment but characteristics of the tumor, particularly benign or maligmant, has not been clarified. In this paper, tomographic patterns of vorious solid (Cystoadenocarcinoma, Granulosa cell tumor and Dysgerminoma) and cystic (Cystoadenoma and Dermoid cyst) tumors are demonstrated. Differential diagnosis to determin malignant or not was still difficult to obtain conclusive result but it was possible to diagnose solid one or not by the method