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The apoptotical effect of nitric oxide on effector apoptotical caspase 3 in rats' hepatocytes was examined. The experiment was performed on 16 white Wistar female rats divided into two equal groups. The rats from the experimental group received orally L-arginine in a dose of 40 mg/kg b.w. every other day for 2 weeks. The rats from the control group received orally 2 ml of distilled water in the same manner as the experimental group. All the rats were decapitated after 3 weeks of the experiment. After decapitation, specimens from the liver were collected, fixed in 10% formalin, and then embedded in paraffin blocks. Protein caspase 3 on slides was detected using the standard three-step immunohistochemical method. The quantitative evaluation of caspase 3 expression showed that the area occupied by positive caspase 3 reaction in the liver of the experimental group (128.11 µm²±96.54) was comparable to that in the control group (212.18 µm² ±1 16.59) (P=0.25). The dose of L-arginine used was similar to that applied in pregnant women treated for gestosis. The study shows that L-arginine as a donor of exogenous nitric oxide has no an apoptotic effect on rats' hepatocytes.
Estrogens exert protective effects against neurotoxic changes induced by over-activation of ionotrophic glutamate receptors, whereas little is known about their interaction with changes mediated by metabotropic glutamate receptors. We evaluated effects of estrone on quisqualate (QA)-induced toxicity in neuronal cell cultures on 7 and 12 day in vitro (DIV). Twenty four hour exposure to QA (150 µM and 300 µM) significantly decreased cell survival in 7 day old cultures, but the 12 day old cultures were more resistant to its toxicity. DNQX (10 µM), an AMPA/kainate receptor antagonist, partly attenuated the toxic effects of QA, whereas LY 367 385 (100 µM), a selective mGluR1alpha antagonist, completely reversed the above effect. QA did not activate, but suppressed spontaneous caspase-3-like activity. Estrone (100 nM and 500 nM) attenuated QA-mediated neurotoxic effects independently of estrogen receptors, as indicated with ICI 182, 780 and without affecting the caspase-3-like activity. At early stage of development in vitro (7 DIV) toxic effects of QA were more profound and mediated mainly by metabotropic glutamate receptors of group I, whereas later (12 DIV) they were mediated mostly by ionotropic AMPA/kainate receptors. The toxic effects of QA were partly accompanied by anti-apoptotic action against spontaneous caspase-3-like activity, possibly due to modulation of neuronal plasticity.
Physiological process of cell death, apoptosis, plays a beneficial role in organism survival, but in some pathologies, like gastric Helicobacter pylori (Hp) infection, this process may turn against the host organism causing tissue damage. Knowledge of the mechanisms controlling apoptosis may have potential significance in treatment of these pathologic states. Therefore, we sought to determine whether apoptosis induced in the gastric epithelial cells exposed to live Hp involves the alteration in heat shock protein 70 (HSP70) expression and activation of caspase-3 in peroxisome proliferator-activated receptors (PPARg dependent manner). Experiments were performed with KATO III, gastric epithelial cells, exposed to CagA and Vac A positive live Hp , water Hp extracts or Hp culture supernatant over different time periods. Total cellular RNA and proteins were isolated for PCR, western-blot and EMSA studies. Genomic DNA was isolated to analyze apoptosis status. We propose new model of Hp induced HSP70 dependent, caspase-3 executed apoptosis in human gastric epithelium. KATO III cells exposed to Hp , showed an increase in caspase-3 activity accompanied and preceeded by activation of nuclear translocation of PPAR peaking at 48 h of culture. Moreover, heat shock factor 1 (HSF-1) bound up with phosphorylated STAT-3 was unable to activate HSP70 protein synthesis in KATO III exposed to Hp . Lack of protective effect of HSP70, activation of caspase-3 - dependent apoptosis pathway caused by Hp and alteration of the bax/bcl-2 cellular equilibrium led to gastric epithelial cell death. The observed phenomenon might be helpful in understanding of the mechanism of Hp related gastrointestinal tract diseasess, especially gastric cancer.
In this study, we investigated apoptosis induced in human trisomic and diabetic fibroblasts by daunorubicin (DNR) and its derivative, idarubicin (IDA). The cells were incubated with DNR or IDA for 2 h and then cultured in a drug-free medium for a further 2–48 h. The apoptosis in the cultured cell lines was assessed by biochemical analysis. We found that both drugs induced a timedependent loss of mitochondrial membrane potential, and a significant increase in intracellular calcium and caspase-3 activity. Mitochondrial polarization and changes in the level of intracellular calcium were observed during the first 2–6 h after drug treatment. Caspase-3 activation occurred in the late stages of the apoptotic pathway. Our findings also demonstrated that idarubicin was more cytotoxic and more effective than daunorubicin in inducing apoptosis in trisomic and diabetic fibroblasts.
Although genetic factors are a well-known cause of colorectal cancer, environmental factors contribute more to its development. Despite advances in the fields of surgery, radiotherapy and chemotherapy, the cure rates for colon cancer have not substantially improved over the past few decades. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the principal pungent ingredient of hot chili pepper, has exhibited an anti-tumor effect in many cell types. However, the mechanisms responsible for the anti-tumor effect of capsaicin are not yet completely understood. In this study, we investigated whether capsaicin induces apoptosis in colon cancer cell lines. Capsaicin decreased cell viability in a dose-dependent manner in Colo320DM and LoVo cells. In addition, capsaicin produced cell morphology changes and DNA fragmentation, decreased the DNA contents, and induced phosphatidylserine translocation, which is a hallmark of apoptotic cell death. We showed that capsaicin-induced apoptosis is associated with an increase in ROS generation and a disruption of the mitochondrial transmenbrane potential. A possible mechanism of capsaicin-induced apoptosis is the activation of caspase 3, a major apoptosis-executing enzyme. Treatment with capsaicin induced a dramatic increase in caspase 3 activity, as assessed by the cleavage of Ac-DEVD-AMC, a fluorogenic substrate. In conclusion, our results clearly showed that capsaicin induced apoptosis in colon cancer cells. Although the actual mechanisms of capsaicin-induced apoptosis remain uncertain, it may be a beneficial agent for colon cancer treatment and chemoprevention.
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