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EN
The current study was undertaken to elucidate a possible neuroprotective role of dehydroepiandrosterone (DHEA) against the development of Alzheimer's disease in experimental rat model. Alzheimer's disease was produced in young female ovariectomized rats by intraperitoneal administration of AlCl3 (4.2 mg/kg body weight) daily for 12 weeks. Half of these animals also received orally DHEA (250 mg/kg body weight, three times weekly) for 18 weeks. Control groups of animals received either DHAE alone, or no DHEA, or were not ovariectomized. After such treatment the animals were analyzed for oxidative stress biomarkers such as hydrogen peroxide, nitric oxide and malondialdehyde, total antioxidant capacity, reduced glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase activities, antiapoptotic marker Bcl-2 and brain derived neurotrophic factor. Also brain cholinergic markers (acetylcholinesterase and acetylcholine) were determined. The results revealed significant increase in oxidative stress parameters associated with significant decrease in the antioxidant enzyme activities in Al-intoxicated ovariectomized rats. Significant depletion in brain Bcl-2 and brain-derived neurotrophic factor levels were also detected. Moreover, significant elevations in brain acetylcholinesterase activity accompanied with significant reduction in acetylcholine level were recorded. Significant amelioration in all investigated parameters was detected as a result of treatment of Al-intoxicated ovariectomized rats with DHEA. These results were confirmed by histological examination of brain sections. These results clearly indicate a neuroprotective effect of DHEA against Alzheimer's disease.
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Glucose deprivation is a factor evoking endoplasmic reticulum (ER) stress and induction of expression of an oxygen-regulated protein of 150 kDa (ORP150). We studied the effect of inducible overexpression of ORP150 on senescence and apoptosis of human breast carcinoma cells (MCF7) and human skin fibroblasts. We found an inhibitory effect of ORP150 on apoptosis and senescence of MCF7 cells, but not fibroblasts in ER stress conditions. An increased expression of senescence-associated β-galactosidase and acid β-galactosidase activity (biomarkers of cellular senescence) was observed. We suggest that ORP150 induction in cancer cells can promote tumour progression and may be a major cause of their resistance to chemotherapeutics.
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Content available remote Evaluation of Selected Indicators of Apoptosis in Patients with Thyroid Tumors
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The aim of the study. Estimatation of p53 protein and soluble FasL ligand level concentration in serum of patients with benign and malignant primary follicular thyroid tumors as indicators of apoptosis and evaluation of their usefulness for early diagnosis of thyroid tumors.Material and methods. 42 patients were qualified for the study. 28 patients were diagnosed with follicular neoplasm (NF) in preoperative fine-needle biopsy. The final verification was postoperative histopathology. Control group consisted of 14 patients with euthyroid goiter, with no cancerous cells detected in cytologic examination. All patients underwent surgical procedures. Levels of p53 and sFasL were marked on the day of admission, before surgery. Destinationes were made in the serum using the ELISA immunoenzymatic method. Obtained data underwent computer statistical analysis.Results. The analysis revealed significantly higher sFasL and p53 concentration in blood of patients with follicular thyroid cancer in comparison with the control group. Similarly, p53 serum level was significantly higher in case of patients with benign thyroid adenoma than in the control group. Comparison between p53 and sFasL serum level in cases of patients with follicular cancer and follicular adenoma showed statistically higher sFasL blood concentration in cases of patients with follicular cancer; there was no statistically significant connection in case of p53 concentration.Conclusions. 1. sFasL and p53 serum concentration are significantly higher in patients with follicular thyroid cancer than in the control group. 2.The p53 serum concentration is significantly higher in cases of all patients with benign thyroid adenoma than in the control group. There was no such correlation for sFasL concentration. 3. sFasL serum concentration is significantly higher in cases of patients with follicular thyroid cancer than in patients with benign thyroid adenoma. There was no such correlation with serum levels of p53.
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Anti-cancer activities of some pyrano-pyridines have been previously reported. Herein, we investigated anti-proliferative and apoptotic effects of the novel pyrano [3, 2-c] pyridine (P.P, TPM.P, 4-CP.P and 3-NP.P) compounds against MCF-7 breast cancer cells. The MCF-7 cells were cultured in the presence of various concentrations (20-200 μM) of the tested compounds for 3 days and the cell viability was determined by MTT assay. Induction of apoptosis was qualitatively assayed by acridine orange/ethidium bromide (AO/EtBr) staining, DNA fragmentation assay, as well as quantitatively by Annexin V/PI double staining and cell cycle analysis. These compounds inhibited growth and proliferation of the MCF-7 cells in a dose- and time-dependent manner. The IC50 values of P.P, TPM.P, 4-CP.P and 3-NP.P after 24 h of exposure were calculated to be 100±5.0, 180±6.0, 60±4.0 and 140±5.0 μM, respectively. 4-CP.P was determined as the most potent compound and was chosen for further studies. The result of flow cytometric cell cycle analysis indicated an increase in sub-G1 population after 72 h treatment of the cells. Furthermore, this was accompanied by exposure of phosphatidylserine (PS) in the outer cell membrane after time course of treatment with the 4-CP.P. Based on these observations, the pyrano [3, 2-c] pyridines can be regarded as a valuable candidate for further pharmaceutical evaluations.
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Bim is defined as the pro-apoptotic BH3-only protein of the Bcl-2 family, which is a critical sensor and mediator in the mitochondrial-dependent apoptosis. In a previous work, we have cloned a novel transcript of Bim (GenBank accession number: AY305716) from the fetal brain cDNA, which is widely expressed in some carcinoma tissues and normal human tissues. According to the sequence analysis and the newly-defined nomenclature system of Bim isoforms (Adachi et al., 2005, Cell Death Differ 2: 192), we term it BimSs3 according to its characteristic structure. The subcellular location analysis indicated that the fused protein GFP-BimSs3 is distributed in the whole cell, mainly to the nucleus. Overexpression of BimSs3 in HEK293 cells causes apoptosis (28.16 ± 1.55%) compared to the negative control (5.44 ± 2.63%). It also causes cytochrome c release from the mitochondrial fraction to the cytosolic fraction during apoptosis. Western blotting assay indicates the molecular mass of GFP-BimSs3 is approximately 31.0 kDa (GFP: 27 kDa). Hence the open reading frame of BimSs3 may initiate at the second ATG and encodes a 36 amino-acid peptide with BH3 domain.
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Programmed cell death (apoptosis) plays a vital role in the regulation of cellular homeostasis. Because of apoptosis fundamental importance, this process is highly regulated. One important set of factors involved in apoptosis regulation is the Bcl-2 family proteins. Bcl-2 family members form a complex regulatory network that controls cell survival and death in response to different physiological and pathological signals. This family includes both pro- and anti-apoptotic members, and Bax protein (Mol wt 21 kDa) is a major pro-apoptotic factor with multifunctional activity. This review summarizes new data about the main representative of Bcl-2 family – Bax, its structure and mechanism(s) by which this protein modulates apoptosis.
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Content available Blood platelets apoptosis in hemodialyzed patients
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Blood platelet proteome of hemodialyzed uremic patients exhibits significant difference in comparison to the blood platelet proteome of healthy subjects. This alteration is manifested by the presence of high concentrations of low molecular peptides within the whole range of pI. Increased platelet apoptosis has been put forward as a possible cause of this phenomenon (1). The aim of the present research was to assess whether blood platelet populations from hemodialyzed uremic patients exhibit more binding sites for Annexin V (a marker of apoptosis) than control samples from healthy donors. Blood was obtained from uremic patients immediately before and after hemodialysis. At the same time samples from control healthy donors were also collected. Blood was anticoagulated with sodium citrate and was immediately exposed to propidium iodide, fluorescent labeled Annexin V and CD61 antibodies. The samples were incubated for 10 minutes in the dark and next the labeled samples were processed in a BectonDickinson FACScan flow cytofluorymeter. Our preliminary study was performed for 12 hemodialyzed patients, 13nondialyzed uremic patientsand 12 controls. It was found that the blood platelet population of hemodialyzed patients exhibited significantly higher level of fluorescence intensity attributed to Annexin V. Furthermore, this intensity was comparable before and after hemodialysis and was independent on patient age. The results support the hypothesis that blood platelet contact with artificial surfaces during the process of hemodialysys may be partially responsible for triggering blood platelet apoptosis.
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Content available remote Upregulation of GRP78 and caspase-12 in diastolic failing heart
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Background: The endoplasmic reticulum (ER) fulfills multiple cellular functions. Various stimuli can potentially cause ER stress (ERS). ERS is one of the intrinsic apoptosis pathways and apoptosis plays a critical role in hypertension. Glucose regulated protein 78 (GRP78) has been widely used as a marker for ERS and caspase-12 mediated apoptosis was a specific apoptotic pathway of ER. The expression of GRP78 and caspase-12 remains poorly understood in the diastolic heart failure resulting from hypertension. Methods: We used spontaneously hypertensive rats (SHRs) to establish a model of diastolic heart failure, and performed immunohistochemistry, western blot, and real-time PCR to analyze GRP78 and caspase-12. Results: We found that GRP78 and caspase-12 had enhanced expression at protein and mRNA levels. Conclusions: These results suggest that GRP78 and caspase-12 were upregulated in cardiomyocytes and ERS can contribute to cardiac myocyte apoptosis in the diastolic heart failure resulting from hypertension.
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Content available remote Effect of aging on UVC-induced apoptosis of rat splenocytes.
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UVC-induced apoptotic symptoms such as morphological changes, DNA fragmentation, Bcl-2 and Bax protein expression were examined in primary splenocyte cultures from young (3 months) and old (24 months) rats. The activities of AP-1 and CRE transcription factors in UVC-irradiated splenocytes were also assessed. At 24 h after UVC irradiation 40% of cells derived from young rats were found to be apoptotic, which was twice as much as in splenocytes from old rats. Apoptosis in cells from old rats did not give typical symptoms like a "DNA ladder" and Bcl-2 protein downregulation, in contrast to splenocytes from young rats. No AP-1 transcription factor activity was found in UVC-irradiated splenocytes from old animals and only a trace activity in splenocytes from young animals. This indicates that, UVC-induced apoptosis in rat splenocytes is practically AP-1 independent and that cells from old rats are less sensitive to UVC irradiation than splenocytes from young rats.
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This study examined the antiproliferative effects of β-escin (E) in cancer cells. The study showed that E inhibited cancer cells growth in a dose-dependent manner. The flow cytometric analysis revealed an escin-induced increase in the sub-G1 DNA content, which is considered to be a marker of apoptosis. Apoptosis was also confirmed by annexin V staining and DNA fragmentation assay. These effects were associated with increased generation of reactive oxygen species (ROS), caspase-3 activation and decreased mitochondrial membrane potential (MMP). Moreover, escin decreased mitochondrial protein content and mitochondrial fluorescence intensity as well as caused depletion of glutathione (GSH). However, activity of glutathione peroxidase (GPx) and glutathione reductase (GR) was not significantly changed in escin-treated cells. In conclusion, our results demonstrated that E has apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Although the exact mechanism needs to be investigated further, it can be concluded that E may be a useful candidate agent for cancer treatment.
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Purpose: The embryo transfer into the uterus by a transcervical catheter is the final stage of in-vitro fertilization procedure. So far, a little attention has been placed on the impact of embryo transfer procedure on embryo viability. The study was design to measure the morphological changes and apoptosis rate in embryos exposed to embryo transfer in in-vitro conditions. Methods: Morphologically normal rabbit blastocysts were divided randomly into control (48) and experimental (48) groups. The experimental group blastocysts were exposed to embryo transfer in in-vitro conditions. Morphological changes in response to embryo transfer were assessed 5 and 60 minutes after ET. The apoptosis rate was measured one hour after embryo transfer. Results: Morphological changes in response to embryo transfer were more prevalent in the experimental group; 14 shrunken and one collapsed blastocysts in experimental group and only two shrunken blastocysts in the control group. The mean DNA fragmented nucleus index in the experimental group was 37.7 % and was significantly higher than in the control group, 8.1 %. Conclusions: Embryo transfer can trigger both morphological and apoptotic changes in rabbit blastocysts. The local pressure fluctuations during embryo transfer could be a major factor responsible for the above-mentioned changes.
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We have demonstrated for the first time that the steroid metabolite, 2-methoxyestradiol (2-ME) is a powerful growth inhibitor of human osteosarcoma 143 B cell line by pleiotropic mechanisms involving cell cycle arrest at two different points and apoptosis. The ability of 2-ME to inhibit cell cycle at the respective points has been found concentration dependent. 1 μM 2-ME inhibited cell cycle at G1 phase while 10 μM 2-ME caused G2/M cell cycle arrest. As a natural estrogen metabolite 2-ME is expected to perturb the stability of microtubules (MT) in vivo analogously to Taxol - the MT binding anticancer agent. Contrary to 2-ME, Taxol induced accumulation of osteosarcoma cells in G2/M phase of cell cycle only. The presented data strongly suggest two different mechanisms of cytotoxic action of 2-ME at the level of a single cell.
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Ovocystatin is a chicken egg white protein, generally known for its inhibitory activity against cysteine proteases. However, biological activity of ovocystatin does not seem to be well recognized in respect to other possible cellular effects. Our attention has been focused on ovocystatin cytotoxic effects in relation to its influence on actin cytoskeleton organization and apoptosis induction. In vitro studies with human melanoma A375, human cervix HeLa cancer cells and normal human fibroblasts - NHDF were done. Cytotoxic activity of ovocystatin was seen in respect to apoptosis induction - manifested by cell shape changes, phosphatydylserine translocation and actin cytoskeleton reorganization. Normal human fibroblasts have shown lower sensitivity to ovocystatin as compared with human melanoma A375 and human cervix HeLa cancer cells. In conclusion, ovocystatin affects actin cytoskeleton organization and displays proapoptotic activity towards applied cell lines. This implicates its application as a potential anticancer drug. However, its adverse effects on normal cells should be taken into consideration.
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Ulcerative colitis (UC) is a inflammatory disease of large bowel. The amount of people suffering from UC increases from year to year. Pathogenesis of this affection is still not entirely clear. Mechanisms of proliferation and apoptosis in colonocytes in the course of the disease are defectedThe aim of the study was to assess the rate of proliferation and intensity of apoptosis in colonocytes in patients with diagnose UC.Material and methods. Colon pathological samples taken from patients with diagnosed ulceraive colitis were examined. Patients were in both clinical and endoscopic remission and were treated with mesalazin. They were patient of Department of General and Colorectal Surgery. To estimate proliferation index dye with monoclonal antibody against Ki67. To determine apoptosis level immunohistochemistry with antybody against Bax was used.Results. Average Ki-67 in the test group was 42,13%, the largest value amounted to 57% and the lowest of 33%. Average value of Bax was 1.47 and ranged between 0-3. High index of bax appear not only in the bottom of the crypt, but also at their outlet.Conclusions. In ulecerative colitis genetic and immunological disturbances occur despite treatment. Mesalazine acting only on certain routes associated with the UC holds the remission, without, however "the molecular remission". Thus, it appears that the results of our research are another proof of the necessary caution in weaning support treatment.
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Cervical cancer is the fourth leading cause of malignancy-related mortality in women worldwide, and effective advanced-stage therapies are urgently required. Berberine is a quaternary ammonium compound extracted from traditional Chinese medicinal herbs, including Phellodendron spp., with antibacterial and antitumor activities. Matrine, the main active ingredient of Sophora flavescens rhizomes, has not only traditionally described health effects but is also widely used for its anticancer, anti-inflammatory, immunoregulatory, antiviral, and hepatoprotective effects. We investigated the antitumor activities of berberine and matrine against human cervical cancer HeLa and SiHa cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, along with flow cytometry and western blotting analyses, to assess the effect of the compounds on the cellular status and apoptosis- and cell cycle-related proteins. The 24 h half-maximal inhibitory concentrations (IC50) of berberine and matrine were 123.633 ± 4.278 µmol/L and 9.625 ± 0.245 mmol/L against HeLa cells and 105.067 ± 3.745 µmol/L and 8.50 ± 0.23 mmol/L against SiHa cells, respectively. Berberine plus matrine inhibited cancer cell growth and caused cell cycle arrest. We observed an increased stimulation of apoptosis, which was likely mediated by enhanced levels of caspase-3, caspase-9, and B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), as well as decreased Bcl-2 protein expression. Cell cycle arrest in the G1 phase was probably mediated by p21 upregulation and cyclin-dependent kinase (Cdk)-4, Cdk-6, and cyclin D1 suppression. Combination treatment with berberine and matrine effectively inhibited human cervical cancer cell proliferation, most likely by stimulating apoptosis and inducing cell cycle arrest.
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Apoptoza jest to ściśle regulowany, naturalny proces zaprogramowanej śmierci komórek w organizmie wielokomórkowym. Dzięki niemu z organizmu usuwane są zużyte lub uszkodzone komórki. Istnieją dwa podstawowe tory indukcji sygnału do apoptozy zewnętrzny i wewnętrzny. Droga zewnętrzna, której poświęcona jest niniejsza praca - jest to szlak indukowany przez czynniki zewnętrzne pobudzające błonowe receptory, posiadające wewnątrzkomórkową domenę zwaną domeną śmierci (death domain) lub też atakiem limfocytów T cytotoksycznych. Możliwe jest też zainicjowanie apoptozy przez połączenie białek błonowych Fas i FasL w obrębie tej samej komórki W pracy przeanalizowano cechy charakterystyczne niektórych receptorów błonowych, zwanych receptorami śmierci, pobudzanych w przebiegu zewnętrznego toru indukcji sygnału do apoptozy. Receptory, które wzięto pod uwagę to: TNFR-1, CD95/ TRAIL-R1, TRAIL-R2, NGFR.
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Apoptosis is a strictly regulated, natural process of programmed cell death in multicellular organism. Via this process, worn-out or damaged cells are removed from the body. There are two basic paths to signal the induc tion of apoptosis - external and internal. The external path, which this work is devoted to, i s induced by external factors stimulating membrane receptors which have the intracellular dom ain called death domain or cytotoxic T lymphocyte attack. It is also possible to initiat e apoptosis by a combination of membrane proteins Fas and FasL within the same cell. The study analyses the characteristics of some memb rane receptors called death receptors, that are stimulated in the course of an external track of induction signal to apoptosis. Receptors taken into account are: TNFR-1 , CD95 / TRAIL-R1, TRAIL-R2, NGFR
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The effects of specific inhibitors of respiratory chain, FoF1ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidingg, antimycin, myxothiazol), the F1-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.
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Content available remote The DFF40/CAD endonuclease and its role in apoptosis.
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The sequential generation of large-scale DNA fragments followed by internucleosomal chromatin fragmentation is a biochemical hallmark of apoptosis. One of the nucleases primarily responsible for genomic DNA fragmentation during apoptosis is called DNA Fragmentation Factor 40 (DFF40) or Caspase-activated DNase (CAD). DFF40/CAD is a magnesium-dependent endonuclease specific for double stranded DNA that generates double strand breaks with 3'-hydroxyl ends. DFF40/CAD is activated by caspase-3 that cuts the nuclease's inhibitor DFF45/ICAD. The nuclease preferentially attacks chromatin in the internucleosomal linker DNA. However, the nuclease hypersensitive sites can be detected and DFF40/CAD is potentially involved in large-scale DNA fragmentation as well. DFF40/CAD-mediated DNA fragmentation triggers chromatin condensation that is another hallmark of apoptosis.
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Content available remote Direct tumor damage mechanisms of photodynamic therapy.
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Photodynamic therapy (PDT) is a clinically approved and rapidly developing cancer treatment regimen. It is a minimally invasive two-stage procedure that requires administration of a photosensitizing agent followed by illumination of the tumor with visible light usually generated by laser sources. A third component of PDT is molecular oxygen which is required for the most effective antitumor effects. In the presence of the latter, light of an appropriate wavelength excites the photosensitizer thereby producing cytotoxic intermediates that damage cellular structures. PDT has been approved in many countries for the treatment of lung, esophageal, bladder, skin and head and neck cancers. The antitumor effects of this treatment result from the combination of direct tumor cell photodamage, destruction of tumor vasculature and activation of an immune response. The mechanisms of the direct photodamage of tumor cells, the signaling pathways that lead to apoptosis or survival of sublethaly damaged cells, and potential novel strategies of improving the antitumor efficacy of PDT are discussed.
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