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.
SUMMARY The purpose of the present study was to investigate erythrocyte membrane abnormalities in hypertension by means of an electron spin resonance and spin-label technique. The erythrocytes from spontaneously hypertensive rats (SHR) and humans with untreated essential hypertension were examined and compared with their normotensive counterparts, and electron spin resonance spectra were obtained for a fatty spin-label agent (5-nitroxy stearate) incorporated into the erythrocyte membranes. The value of outer hyperfine splitting (2T\) was significantly higher in erythrocytes of SHR and humans with essential hypertension than in erythrocytes of normotensive controls (at 37 °C: SHR, 56.14 ± 0.51 gauss [G], n = 8; Wistar-Kyoto rats, 52.22 ±0.86 G, n = 4, p<0.01; humans with essential hypertension, 56.94 ± 0.27 G, n = 11; normotensive subjects, 55.44 ± 0.36 G, n = 8, p<0.01). The order parameter (5) was also increased in the hypertensive rats and humans compared to their respective normotensive controls. When calcium was loaded to erythrocytes with calcium ionophore A23187 (0.9 fiM) and CaCl 2 (1.0 mM), the parameters of the spectra were increased. These changes were more prominent in the hypertensive groups than in the normotensive controls. These results revealed that the erythrocyte membranes of the hypertensive subjects tolerated different spin motions than those of the normotensive controls in the electron spin resonance study and that membrane fluidity might be decreased in hypertension. Additionally, calcium loading to erythrocytes caused the reduction of membrane fluidity. Therefore, it is suggested that an abnormality of calcium handling at the cellular level might affect physical properties of the biomembranes in hypertension. 1 " 5 It seems likely that generalized abnormalities of the membranes could be involved not only in vascular smooth muscle cells but also in circulating blood cells. Erythrocytes are widely studied because they are a
Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan degradation in the placenta has been implicated in the prevention of the allogeneic fetus rejection [Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, and Mellor (1998) Science 281, 1191-1193]. To determine how IDO is associated with the development of the fetus and placenta, the time course of IDO expression (tryptophan-degrading activity, IDO protein and IDO mRNA) in the embryonic and extra-embryonic tissues as well as maternal tissues of mice was examined. A high tryptophan-degrading activity was detected in early concepti on days 6.5 and 7.5, whereas IDO protein and its mRNA were not expressed during early gestation, but appeared 2-3 days later, lasted for about 3 days and declined rapidly thereafter. The expression of IDO basically coincided with the formation of the placenta. On the contrary, the early tryptophan-degrading activity was due to gene expression of tryptophan 2,3-dioxygenase (TDO), as shown by Northern and Western analysis. These findings indicate that IDO is transiently expressed in the placenta but that the expression does not last until birth, and that the IDO expression is preceded by expression of another tryptophan-degrading enzyme, TDO, in the maternal and/or embryonic tissues in early concepti.
Hepatitis C virus (HCV) causes persistent infection inhepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused upregulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.Hepatitis C virus (HCV) 1 causes the persistent infection chronic hepatitis in most infected patients. This disorder eventually progresses to cirrhosis and hepatocellular carcinoma (HCC). Numerous studies have provided evidence supporting a link between chronic HCV infection and HCC (1, 2). However, exactly how HCV infection could be directly involved in the development of HCC remains unclear because of the lack of an efficient in vitro infection system.HCV is a member of the Flaviviridae family and has a positive-strand RNA genome (ϳ9.6 kb). Viral proteins are synthesized as a single polyprotein, which is then cleaved into structural (core, E1, and E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. In vitro expression of HCV core protein reportedly influences cell growth regulation, and the protein can interact with the cytoplasmic tail of a lymphotoxin- receptor (3), the death domain of tumor necrosis factor receptor-1 (4), and NF-B (5), resulting in enhancement or inhibition of Fas-and tumor necrosis factor-␣-mediated apoptosis. HCV structural protein inhibits Fas-mediated apoptosis by inhibiting cytochrome c release from mitochondria in mice (6). Cellular transformation has been shown to be caused by HCV core protein in the presence of the ras gene (7,8) and by loss of function of LZIP (9) and the presence of STAT3 (signal transducer and activator of transcription-3) (10). NS3 (11) and NS4B (12) proteins reportedly display tumorigenicity in the presence of ras. These results indicate that expression of individual HCV proteins does not cause cellular transformation in vitro. HCV core protein transgenic mice reportedly show induced steatosis and, after 16 months of age, develop HCC (13). In contrast, HCV structural protein transgenic mice do not display neoplastic or cancerous lesions in the liver by 20 months of age (14). Moreover, conditional expression of an HCV structural protein region (nucleotides 294 -3435) causes hepatic injury in transgenic mice (15), but HCC is not observed by at least 16 months of age (data not shown). The frequency of HCC occurrence is reportedly higher in full-...
Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan degradation in the placenta has been implicated in the prevention of the allogeneic fetus rejection [Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, and Mellor (1998) Science 281, 1191-1193]. To determine how IDO is associated with the development of the fetus and placenta, the time course of IDO expression (tryptophan-degrading activity, IDO protein and IDO mRNA) in the embryonic and extra-embryonic tissues as well as maternal tissues of mice was examined. A high tryptophan-degrading activity was detected in early concepti on days 6.5 and 7.5, whereas IDO protein and its mRNA were not expressed during early gestation, but appeared 2-3 days later, lasted for about 3 days and declined rapidly thereafter. The expression of IDO basically coincided with the formation of the placenta. On the contrary, the early tryptophan-degrading activity was due to gene expression of tryptophan 2,3-dioxygenase (TDO), as shown by Northern and Western analysis. These findings indicate that IDO is transiently expressed in the placenta but that the expression does not last until birth, and that the IDO expression is preceded by expression of another tryptophan-degrading enzyme, TDO, in the maternal and/or embryonic tissues in early concepti.
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