ABSTRACT. Westudied the effects of bafilomycin Ai, a potent and specific inhibitor of vacuolar H+ ATPase (V-ATPase), on the process of autophagy in rat hepatoma cell line, H-4-II-E cells. To induce autophagy, cells were transferred from Dulbecco's modified Eagle medium containing 12% fetal calf serum into Hanks' balanced salt solution. When bafilomycin Ai was added to Hanks' balanced salt solution, endogenous protein degradation was strongly inhibited and numerous autophagosomes accumulated in H-4-II-E cells, whereas autolysosomes decreased in number. Acid phosphatase activity was not detected in the autophagosomes which accumulated in the presence of bafilomycin Ai, suggesting that fusion between autophagosomes and lysosomes was disturbed by this drug. Inhibition of the fusion was reversible, and the autophagosomes changed into autolysosomes after the removal of the inhibitor. Bafilomycin Ai also prevented the appearance of endocytosed HRPin autophagic vacuoles. These results suggested that acidification of the lumenal space of autophagosomesor lysosomes by V-ATPase is important for the fusion between autophagosomes and lysosomes.Autophagy is one of the main pathways of the degradation of the endogenous proteins and organella (4, ll, 32). In the process of autophagy, membrane structures called isolation membranesor phagophores appear, segregate parts of the cytoplasm, and form autophagosomes. The newly formed autophagosomes (early autophagosomes) fuse with endosomes or prelysosomes, and becomea more advanced stage of autophagosomes (late autophagosomes or amphisomes) of acidic luminal pH (2, 1 1, 12). The autophagosomes then acquire hydrolytic enzymes by fusion with lysosomes, and are transformed into mature autolysosomes in which degradation of the content proceeds (10, ll, 40). Vacuolar H+ ATPase (V-ATPase) is localized in organelles of the central vacuolar system such as coated vesicles, endosomes, lysosomes, chromaffin granules, and Golgi apparatus, and plays an important role by maintaining the acidic environment in these compartments (for review, see Mellman et al). Bafilomycin Ai, a macrolide antibiotic isolated from Streptomyces sp., is a highly specific inhibitor of the V-ATPase (5, 14, 38). Inhibition by bafilomycin Ai occurs through binding to the membrane-spanning pore forming domain of the enzyme (7, 42). Bafilomycin Ai was also effective on living cells when added extracellularly (36, 41). Therefore it is a powerful tool to study the role of V-ATPase and vacuolar pH. Using this drug, we have previously reported that V-ATPase is essential for acidifying the lumen of lysosomes and subsequent protein degradation of endocytosed epidermal growth factor (EGF) in lysosomes of cultured cells (41) and phagocytosed rod outer segmentsin phagolysosomesof retinal pigmentepithelial cells (9).In this study, to clarify the roles of V-ATPasein autophagy, we studied the effects of bafilomycin Ax on the process of autophagy in rat hepatoma cell line, H-

A crossbow is a bow that shoots an arrow when a gun-like trigger is pulled. Deaths caused by accidental crossbow shootings are extremely rare. Here we describe an autopsy case of a penetrating wound to the left cerebral hemisphere caused by an accidental shooting with a crossbow. A man in his early 60s who lived with his wife and had used crossbows for 20 years as his hobby was found one early morning in the shed of his house, collapsed and bleeding from the head and neck. He was taken to a hospital and died after approximately 3 days of conservative treatment. At autopsy, a penetrating wound between the upper part of the left anterior neck and the left frontoparietal region was evident. Traumatic intracerebral hematoma was observed in the left frontal lobe, and severe traumatic subarachnoid hemorrhage was present throughout the brain. Cerebral contusion and hematoma without any organization were noted around the penetration. The cause of death was determined to be cerebral contusion and intracerebral hematoma due to the penetrating wound by the crossbow arrow. He was probably trying to load an arrow into the crossbow by placing it on the floor, pointing upward, and made a mistake in its operation that resulted in the shooting of the arrow. This case is unique because it was a rare accidental death caused by a crossbow arrow, and a detailed histopathological examination was performed.

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F i g u r e 1 A. Ch e s t r a d i o g r a p h t a k e n a t t h e t i me o f a d mi ss i o n s h o we d d i f f u s e g r o u n d -g l a s s o p a c i t y o n b o t h l u n g s .F i g u r e 1 B . No n c o n t r a s t -e n h a n c e d CT s c a n s h o we d a r i g h t a d r e n a l ma s s wi t h r e t r o p e r i t o n e a l h e ma t o ma . * , r i g h t a d r en a l ma s s ; a r r o wh e a d s , r e t r o p e r i t o n e a l h e ma t o ma . (Fig. 1A). Abdominal computed tomography (CT) showed a hyper-enhancing right adrenal mass with retroperitoneal hematoma (Fig. 1B = non contrastenhanced CT, Fig. 1C, D= serial images of an arterial phase contrast-enhanced CT). The serum and urine catecholamine levels were markedly elevated. showed uptake at the right supra-renal region (Fig. 1E). Based on these findings, the diagnosis of ruptured right adrenal pheochromocytoma was made. Volume supplementation and intravenous/intraoral administration of α-adrenergic blocking agents were continued. The pulmonary edema and dyspnea resolved rapidly without using cardiotonic or diuretic drugs. The patient subsequently underwent surgery. *

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