We have recently developed a simple method of plasma free DNA detection, which enables us to distinguish between apoptotic and genomic (necrotic) DNA. After applying this method to the critically ill, we revealed apoptotic DNA on the day of admission to be higher than later when multiple-organ failure developed. Moreover, apoptotic DNA contributed to total plasma DNA much more than DNA from necrotic cells and its increase predicted future development of multiple-organ failure and death.
The pattern and signal transduction of neuronal apoptosis in the brain of the silk moth, Bombyx mori, during postembryonic life, were characterized. Peak numbers of apoptotic neurons were detected in 4 day old 4th instar larvae, 9 day old 5th instar larvae and 4 day old pupae, indicating three waves of neuronal apoptosis during postembryonic development. Most of the apoptotic neurons were in the lateral portions of the brain. No apoptotic neurons were detected in 1 day old 1st instar larvae or in 7 day old pupae to 1 day old adults. Injection of 20-hydroxyecdysone (20E) into larvae resulted in a substantial increase in the brain in both neuronal apoptosis and cleavage of procaspases-8 and -3 into caspases-8 and -3. However, the injection of larvae with actinomycin D or cycloheximide inhibited death of pre-apoptotic neurons. Both the cleavage of procaspases-8 and -3 and death of pre-apoptotic neurons were inhibited by a general caspase inhibitor and caspase-8 and -3 inhibitors injected into larvae. These results suggest that 20E triggered the synthesis of a new protein that, in turn, induces cleavage of procaspases-8 and -3 into caspases-8 and -3. These caspases are prerequisites for neuronal apoptosis in postembryonic brains.
Apoptosis was induced by treatment of HL-60 cells with C2-ceramide. Apoptotic damage of DNA was detected according to the sub-Gl peak on a flow cytometer, according to the typical morphology and according to the DNA fragmentation "ladder" after gel electrophoresis. It was shown that the apoptotic cleavage followed after G1 blockade of the cell cycle. A high correlation coefficient (rs=0.957) was found between the percentages of G1 blocked cells and apoptotic cells. This high correlation together with the appearance of the sub-Gl peak suggests that the G1 blocked HL-60 cells were subject to apoptotic death. It is deduced that the mechanisms leading to G1 blockade of the cell cycle and activation of apoptosis in HL-60 cells are interconnected.
The effect of phagocytosis of living bacteria on apoptotic DNA changes was examined in pig leukocytes in relation to immune system maturation. Blood samples of pigs (aged 6, 12 and 18 weeks) were cultivated with a suspension of bacterial cells Salmonella typhimurium LB 5000 at 37 °C. In the experimental groups, killed bacteria and microspheric particles were used to detect the influence of the phagocytic process. Phagocytic activity and index were determined in each sample by means of microspheric particles. The ability to kill engulfed microbes (bactericidal capacity) was estimated from the decrease in bacterial colony-forming units (CFU). Samples of cultured cells were taken for DNA analysis at given intervals. DNA ladder assay was used for qualitative apoptotic DNA break detection and the TUNEL AP test was employed for quantification of apoptosis. In 18-week-old animals, spontaneous DNA degradation was observed in the control group without phagocytosis after 8 h. In contrast, cells cultivated with microspheric particles or killed bacteria became apoptotic after 4 h. The rate of apoptotic DNA degradation was decreased in the group exposed to living bacteria. This prolonged survival of phagocytes was also detected in 12-week-old animals, but not at 6 weeks of age. These findings were supported by the ability of phagocytes in 6-week-old animals to engulf microbes, but their killing (bactericidal) ability was significantly decreased in comparison with other stages of immune system maturation. These results suggest that the process of phagocytosis itself is accompanied by activation of the apoptotic program in phagocytic cells of the pig immune system, but the presence of phagocyted living bacteria can delay this activation. The prolonged survival of short-lived cells was only observed in later phases of immune system maturation., E. Matalová, A. Španová, F. Kovářů., and Obsahuje bibliografii