| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1992: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1991: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Rat liver peroxisomes were markedly proliferated by administration of dioctyl phthalate (DEHP) for 2 weeks. When the animals were fed on normal diet for a week further, the number and size of the peroxisomes recovered to normal. When leupeptin was injected into these animals,there was a marked accumulation of autophagosomes in the hepatocytes. Using this as an experimental model,the early stage of the autophagosome formation was analyzed byelectron microscopy and immunocytochemistry. Twenty minutes after the injection,isolation membranes surrounding the target organelles appeared. They were characterized by double layers with a narrow cisternal space and were sometimes continuous with the rough ER. Forty minutes after leupeptin injection,the lumen of the isolation membrane was enlarged and the inner membrane attached to the entrapped material. Enzyme cytochemical staining showed that the isolation membranes were negative for Golgi and lysosomal enzymes,but were strongly positive for an ER marker,glucose-6-phosphatase (G6Pase). The enlarged cisternae of the isolation membranes of the early autophagic vacuoles were in part positive for this enzyme,but gradually became negative with time. Similarly,the G6Pase activity was lost when the inner membrane was degraded. Immunocytochemical staining for ER marker enzymes showed positive reaction in the isolation membranes. After immunostaining for cathepsins and lysosomal membrane glycoprotein,the isolation membranes were consistently negative for these antigens. Forty to 60 minutes after leupeptin injection,reaction-negative autophagic vacuoles were fusing with reaction-positive lysosomal compartments. The results suggest 1) that the isolation membranes enclosing the target organelles are derived from the ER and 2) the early autophagosomes lack lysosomal enzymes and later they gain the enzymes and consequently are converted to autophagolysosomes in which the trapped organelles are degraded.
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