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In this study the contribution of the ERK1/2 pathway to sphingosine-induced death and morphological changes of the actin cytoskeleton in glioma C6 cells was investigated. Surprisingly, the level of ERK1/2 phosphorylation does not change after incubation of cells with sphingosine. Despite this, sphingosine induces rounding and detachment of cells without formation of apoptotic bodies. To shed light on this process, a specific inhibitor of ERK1/2 phosphorylation, U0126, was used. Cells incubated simultaneously with sphingosine and U0126 not only detached, but also exhibited formation of apoptotic-like blebs. These data suggest that during sphingosine-induced glioma C6 cell death apoptotic blebbing is dependent on ERK1/2 signalling and occurs only when ERK1/2 activity is decreased or abolished.
Influence of melittin on viability and integrity of cell membrane on grade IV glioma. The grade IV glioma is one of the malignant human tumours. Today there is no effective treatment for this type of cancer. Alternative methods are sought-after in glioma treatment, and lately melittin has been found to be useful in anticancer therapy. The aim of the study was to investigate the effect of melittin on the viability and the integrity of cell membranes of the grade IV glioma cells. The U87 glioma line cells were treated of melittin in increasing concentrations (5, 10, 15, 20 and 50 µg/mL) and incubated for 24 hours. After incubation, the tests were performed in order to investigate the cell morphology, cell viability, membrane integrity and the way of cell death. The results have shown the devastating effect of melittin on the glioma cells. The melittin causes disintegration of cell membranes and induces cell death by apoptosis and less by necrosis.
In this review we summarize the present status of our knowledge on the enzymes in­volved in the extracellular metabolism of nucleotides and the receptors involved in nucleotide signalling. We focus on the mechanism of the ATP and ADP signalling path­ways in glioma C6, representative of the type of nonexcitable cells. In these cells, ATP acts on the P2Y2 receptor coupled to phospholipase C, whereas ADP on two distinct P2Y receptors: P2Y1 and P2Y12. The former is linked to phospholipase C and the lat­ter is negatively coupled to adenylyl cyclase. The possible cross-talk between the ATP-, ADP- and adenosine-induced pathways, leading to simultaneous regulation of inositol 1,4,5-trisphosphate and cAMP mediated signalling, is discussed.
We assessed the rate of release of a model nucleoside (adenosine, 5%, w/w) from nine different lactide-glycolide or lactide-caprolactone polymers. The polymer discs were eluted every second day with an artificial cerebrospinal fluid at the elution rate roughly approximating the brain extracellular fluid formation rate. Adenosine in eluate samples was assayed by HPLC. Three polymers exhibited a relatively constant release of adenosine for over four weeks, resulting in micromolar concentrations of nucleoside in the eluate. This points to the neccessity of further development of polymers of this types as intracerebral nucleoside delivery systems for local treatment of brain tumors.
Embedment-free electron microscopy (EFEM) is a new method which allows the visualisation of cytoskeleton in whole-mounted cells. In this study we employed EFEM to investigate the structure of cellular scaffolds in glioma C6 cell line. The cells were extracted with Triton X-100 that dissolves phospholipids in the membranes and removes most of cytoplasmic soluble proteins. The DNA and nuclear histones were removed with DNase I and high-salt buffer, respectively. The remaining cellular frameworks were temporary embedded in diethylene glycol distearate (DGD), sectioned and observed in transmission and scanning electron microscope after the removal of DGD. The predominant structure was the extensive meshwork of 10-20 nm filaments in the cytoplasm (cytomatrix) and 15-30 nm filaments in the nucleus (nuclear matrix). The 5 nm filaments, presumably corresponding to the actin filaments, were present in the cytomatrix, but not in the nuclear matrix. Moreover, the ultrathin (3 nm) filaments, connecting other cytoskeletal components were detected. Those are possibly identical with the previously described plectin filaments. For the first time we report the occurrence of ultrathin filaments in the nuclear matrix. Thus, in a addition to the well known cytoskeletal components (microtubules, intermediate filaments, actin microfilaments) EFEM showed a new type of filaments (the ultrathin filaments) in the cytomatrix and nuclear matrix. Further immunocytochemical studies are needed to determine the biochemical identity of the filaments observed in EFEM.
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