During apoptotic execution, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert highly condensed form that is fragmented and packaged into apoptotic bodies. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cellfree system and time-lapse imaging. We demonstrated that the population of nuclei undergoing apoptosis in vitro appears to follow a reproducible program of nuclear condensation, suggesting the existence of an ordered biochemical pathway. This enabled us to define three stages of apoptotic chromatin condensation: Stage 1 ring condensation; Stage 2 necklace condensation; and Stage 3 nuclear collapse/disassembly. Electron microscopy revealed that neither chromatin nor detectible subnuclear structures were present inside the stage 1 ring-condensed structures. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectible DNase activity. However, DNase(s) were required for stage 2 necklace condensation. Finally, we demonstrated that hydrolysable ATP is required for stage 3 nuclear collapse/disassembly. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis.

Cryptococcus neoformans grown on culture media was observed by the freeze-etching technique. In the capsule, short fibrils were seen when freezeetched. This organism was unique in the appearance of the cell wall, which showed two strata. The outer one was dense with particles of about 20 nm in diameter, whereas the inner one was sparse in particles. The appearance of the cell membrane of this organism differed distinctly depending on the culture media. When grown on glycerol medium, the cell membrane possessed, as do other yeasts, clear but somewhat longer and curved invaginations. The membrane of cells grown on nonglycerol medium exhibited, however, only a few invaginations of irregular shape. Instead, characteristically of this organism, the cell membrane showed round depressions of 40 to 200 nm in diameter which were the surface view of the paramural bodies. In cross-fractured cells, both types of paramural bodies were found. Some of them contained a single vesicle of about 50 nm in diameter. These seem to play a role in secreting the cytoplasmic vesicles. Data suggesting the existence of multivesicular bodies in the cytoplasm and of multivesicular lomasomes were also obtained. Some of the baglike paramural bodies showed multilayered membrane. These are thought to be plasmalemmasomes. This organism was similar to other yeasts reported in other respects.

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Cryptococcus neoformans grown in the parasitic state was observed by the freeze-etching technique and was compared with that grown on culture media. Unlike other yeasts, this organism grown in vivo is very often devoid of the "ordinary" invaginations. The membrane of the cell grown in vivo was almost free from concavity and convexity except for many round depressions which represent the surface view of paramural bodies. Some of the paramural bodies were found to be multivesicular systems. Most were spherical invaginations containing a single vesicle or its ghost remaining after secretion of the vesicles. In clear contrast to the cell grown in vitro, the in vivo cell contained a great number of vesicles in the cytoplasm. These seemed to show high-secretion activity in C. neoformans grown in the parasitic state. On transfer from in vitro to in vivo, this organism enlarged the cell wall, capsule, and cell body. The appearance of a large vacuole, accumulation of storage organelles, and the existence of rodlike structures, seemingly lipid deposits, were also noted in the cytoplasm of the cell grown in vivo. The meaning of these results as well as the mode of capsular production are discussed.

Alpha-lipoic acid has been shown to possess cancer-cell-killing activity via activation of the apoptosis pathway. In this study, the cytotoxic activities of alpha-lipoic and dihydro-alpha-lipoic acid were compared in HL-60 cells. The cell-killing activity of dihydro-alpha-lipoic acid was higher than that of alpha-lipoic acid. Both alpha-lipoic and dihydro-alpha-lipoic acid induced caspase-3 cleavage and internucleosomal DNA fragmentation in treated cells. On the other hand, apparent necrotic or late-stage apoptotic cell populations could be detected in dihydro-alpha-lipoic acid cells but not in those treated with alpha-lipoic acid. Moreover, dihydro-alpha-lipoic acid, but not alpha-lipoic acid, induced marked mitochondrial permeability transition. Antioxidants could not prevent dihydro-alpha-lipoic- or alpha-lipoic-acid-induced cell death. In addition, dihydro-alpha-lipoic and alpha-lipoic acid did not up-regulate cellular reactive oxygen level. These results indicated that dihydro-alpha-lipoic acid exerts more potent cytotoxicity than alpha-lipoic acid through different cytotoxic actions.