RESUMO
Extracellular and intraneuronal formation of amyloid-beta (Abeta) deposits have been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of Abeta neurotoxicity is not completely understood. Previous studies suggest that binding of Abeta with a number of targets have deleterious effects on cellular functions. It has been shown that Abeta directly interacted with intracellular protein ERAB (endoplasmic reticulum amyloid beta-peptide-binding protein) also known as ABAD (Abeta-binding alcohol dehydrogenase) resulting in mitochondrial dysfunction and cell death. In the present study we have identified another mitochondrial enzyme, ND3 of the human complex I, that binds to Abeta1-42 by the screening of a human brain cDNA library expressed on M13 phage. Our results indicated a strong interaction between Abeta and a phage-displayed 25 amino acid long peptide TTNLPLMVMSSLLLIIILALSLAYE corresponding to C-terminal peptide domain of NADH dehydrogenase, subunit 3 (MTND3) encoded by mitochondrial DNA (mtDNA). This interaction may explain, in part, the inhibition of complex I activity in astrocytes and neurons in the presence of Abeta, described recently. To our knowledge, the present study is the first demonstration of interaction between Abeta and one of the subunits of the human complex I.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Biblioteca Gênica , Testes Genéticos/métodos , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Southern Blotting/métodos , Fragmentação do DNA/fisiologia , Complexo I de Transporte de Elétrons , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Reação em Cadeia da Polimerase/métodosRESUMO
INTRODUCTION/PURPOSE: The effect of a single bout of intensive exercise on apoptosis of rat neutrophils and the possible prevention by glutamine administration was examined. The experiments were performed in sexually immature and sexually mature male rats as to examine the possible involvement of sexual maturation in the effect of exercise. METHODS: Exercise was carried out on a treadmill for 1 h before rats were killed by decapitation. Aqueous solution of glutamine was given by gavage (1 g.kg-1 body weight), 1 h before exercise. Neutrophils were obtained by intraperitoneal lavage with phosphate-buffered saline (PBS), 4 h after injection of oyster glycogen solution. The cells were then analyzed for apoptosis by flow cytometry and fluorescence microscopy. Pro- and antiapoptotic gene expression was evaluated by reverse transcriptase chain reaction (RT-PCR). RESULTS: Neutrophils obtained from immature and mature exercised rats showed an increase in DNA fragmentation, chromatin condensation, and phosphatidylserine externalization. This suggests that all neutrophils suffered apoptosis. To study the possible mechanism involved, the production of reactive oxygen metabolites, expression of genes involved in apoptosis and mitochondrial transmembrane potential were examined. Acute exercise raised reactive oxygen metabolites production by neutrophils. Exercise did not change the expression of antiapoptotic (bcl-xL) and apoptotic (bax and bcl-xS) genes in neutrophils from immature rats but caused a significant increase of bax and bcl-xS expression and provoked a significant decrease of bcl-xL expression in cells from mature rats. Exercise also induced a marked loss of mitochondrial depolarization in neutrophils. Oral glutamine supplementation partially prevented the exercise-induced apoptosis in neutrophils from sexually immature and mature rats. CONCLUSION: The protective effect of glutamine on neutrophil apoptosis induced by acute exercise possibly occurs by preservation of mitochondrial function.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Glutamina/administração & dosagem , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Condicionamento Físico Animal/fisiologia , Administração Oral , Animais , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Masculino , Ratos , Ratos Wistar , Valores de ReferênciaRESUMO
CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on "reactive oxygen species".
Assuntos
Apoptose/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Naftoquinonas/farmacologia , Naftoquinonas/toxicidade , Animais , Apoptose/fisiologia , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/patologia , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/patologia , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/patologia , Membranas Intracelulares/ultraestrutura , Masculino , Microscopia Eletrônica , Microvilosidades/efeitos dos fármacos , Microvilosidades/patologia , Microvilosidades/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Ratos , Ratos WistarRESUMO
CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species. (AU)
Assuntos
Humanos , Masculino , RESEARCH SUPPORT, NON-U.S. GOVT , Apoptose/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Naftoquinonas/farmacologia , Naftoquinonas/toxicidade , Apoptose/fisiologia , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/patologia , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/patologia , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/patologia , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica , Microvilosidades/efeitos dos fármacos , Microvilosidades/patologia , Microvilosidades/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Ratos , Ratos WistarRESUMO
CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species.
Assuntos
Humanos , Masculino , Apoptose/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Naftoquinonas/farmacologia , Naftoquinonas/toxicidade , Apoptose/fisiologia , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/patologia , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/patologia , Extensões da Superfície Celular/ultraestrutura , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Microscopia Eletrônica , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/patologia , Membranas Intracelulares/ultraestrutura , Microvilosidades/efeitos dos fármacos , Microvilosidades/patologia , Microvilosidades/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Peróxido de Hidrogênio/metabolismo , Ratos , Ratos Wistar , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestruturaRESUMO
Polyunsaturated fatty acids (PUFAs) can influence tumor growth and migration, both in vitro and in vivo. The PUFA gamma-linolenic acid (GLA) has been reported to improve the poor prognosis associated with human gliomas, although its effects at sublethal concentrations on residual cells postsurgery are poorly understood. The study investigated the effects sublethal PUFA doses (90 or 150 microM) may have on rat C6 glioma cell energy metabolism, since an adequate energy supply is essential for cell proliferation, migration, and apoptosis. Of note was the identification of mitochondrial heterogeneity in relation to the mitochondrial membrane potential (MMP), which has been suggested but unproven in previous studies. GLA and eicosapentaenoic acid (EPA) caused significant changes in cellular fatty acid composition and increased the percentage of cells with a low MMP after a 96-h exposure period. The presence of PUFAs inhibited C6 cell proliferation and migration, although apoptosis was not induced. The protein expression and activity of glucose-6-phosphate dehydrogenase was increased after 96-h incubation with 90 microM GLA and EPA and would allow redox regulation through increased NADPH production, permitting the maintenance of adequate intracellular reduced glutathione concentrations and limiting rates of lipid peroxidation and reactive oxygen species generation. Neither NADP(+)-isocitrate dehydrogenase nor NADP(+)-malate dehydrogenase activity responded to PUFAs, suggesting it is glucose-6-phosphate dehydrogenase that is the principal source of NADPH in C6 cells. These data compliment studies showing that higher concentrations of GLA induced glioma cell death and tumor regression and suggest that GLA treatment could be useful for the inhibition of residual cell proliferation and migration after surgical removal of the tumor mass.
Assuntos
Etídio/análogos & derivados , Ácidos Graxos Insaturados/farmacologia , Glioma/patologia , Glucosefosfato Desidrogenase/metabolismo , Animais , Anexina A5/metabolismo , Benzimidazóis/metabolismo , Western Blotting , Carbocianinas/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas/metabolismo , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Etídio/metabolismo , Imunofluorescência/métodos , Corantes Fluorescentes/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glioma/metabolismo , Potenciais da Membrana/fisiologia , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Mitose/fisiologia , Ratos , Fatores de TempoRESUMO
The Taenia crassiceps cysticercus is a cestode that naturally and experimentally infects rodents in which it reproduces by budding. In the laboratory, a persistent cellular immunosuppression with a concomitant increasing load of parasites has been observed in experimentally infected BALB/cAnN mice. In this study, enhanced apoptosis was found in spleen cells from 30-day infected mice with a typical "ladder-patterned" DNA fragmentation and an increase in phosphatidylserine expression. A characteristic poly-(ADP-ribose) polymerase cleavage indicates that this cell death is caspase-mediated. Apoptosis was detected in the CD4(+) and CD19(+) splenocytes of infected mice after in vitro stimulation with cysticercal antigens. Considering previous results on the crucial role that CD4(+) T cells play in controlling the extent of infection, apoptosis in this T-lymphocyte subpopulation induced by T. crassiceps cysticerci could be responsible for the immunosuppression that underlies parasite success.
Assuntos
Antígenos CD19/análise , Apoptose , Linfócitos T CD4-Positivos/imunologia , Baço/imunologia , Teníase/imunologia , Animais , Antígenos de Helmintos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Caspases/metabolismo , Células Cultivadas , Fragmentação do DNA/fisiologia , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilserinas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Baço/citologia , Taenia/classificação , Taenia/crescimento & desenvolvimento , Teníase/patologiaRESUMO
Investigou-se o efeito do colesterol em enterócitos fetais e na linhagem IEC-6. Para isso, analisou-se a composição dos ácidos graxos, a atividade da citrato sintase e a proliferação dessas células na presença de colesterol. A expressão da HMG-CoA redutase nos intestinos fetais, células IEC-6 e em quatro segmentos intestinais de ratos adultos: duodeno, jejuno, íleo e instestino grosso foi determinada por RT-PCR. Estudou-se a indução da fragmentação do DNA e condensação da cromatina na linhagem IEC-6 pelo colesterol, através de citometria de fluxo e microscopia de fluorescência utilizandoo Hoescht 33342. Avaliou-se o efeito do colesterol na expressão da HMG-CoA redutase e PPAR . Também, o efeito de drogas...
Assuntos
Animais , Ratos , Colesterol , Enterócitos , Fragmentação do DNA/fisiologia , Intestinos , Linhagem Celular , Contagem de Células/métodos , Citometria de Fluxo , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Recombinant human erythropoietin (rHuEPO) induces endothelial cell growth and angiogenesis in vitro. The mechanisms are unknown. Because an increase in endothelial cell survival could play a role in this process, we examined the effect of rHuEPO on lipopolysaccharide (LPS)-induced apoptosis in bovine pulmonary artery endothelial cells (BPAECs). METHODS: Four groups of cells were studied. The first group was preincubated in serum-free medium followed by treatment with LPS. The second group was preincubated with rHuEPO followed by LPS. The third group was treated with only rHuEPO. Control cells were cultured in the absence of rHuEPO and LPS. Apoptosis was determined by flow cytometric DNA analysis, propidium iodide staining, cellular DNA fragmentation by ELISA, and gel electrophoresis. RESULTS: LPS-treated cells showed an increase in hypodiploid DNA (36.4 +/- 6.1%) compared with controls (9.8 +/- 3.3%, P < 0.001). Preincubation with rHuEPO decreased this effect to 14.7 +/- 5.1% (P < 0.001). Apoptosis determined by propidium iodide was observed in 33 +/- 8% of LPS-treated cells, but in only 9 +/- 3% of cells preincubated with rHuEPO cells (P < 0.001). Similarly, DNA fragmentation was decreased in rHuEPO pretreated cells compared with LPS alone (0.155 OD +/- 0.02 vs. 0.538 +/- 0.09 OD, P < 0.001). DNA breakdown was observed in only LPS-treated cells. CONCLUSIONS: These results suggest that rHuEPO prevents LPS-induced apoptosis in endothelial cells. This protective effect could be an important factor in the action of rHuEPO on vascular endothelium.