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Lymphocytes were obtained by heart-punction from rats bearing Morris hepatoma. In the short term, 18-hour cultures of these lymphocytes exhibited a significantly higher amount of apoptotic cells than lymphocyte cultures from the healthy, control animals. Neuraminidase, injected into the caudal vein of the rats with Morris hepatoma, caused a marked lowering in the amount of apoptotic blood-lymphocytes and an elevation of the amount of viable cells. The possible mechanism of neuraminidase preventing the apoptosis of blood-circulating lymphocytes in tumour hosts is discussed herein.
Monomeric (G), total (T) and filamentous (F) actin and the state of actin polymerisa­tion (F:G) were determined and actin filaments were visualized in hepatoma Morris 5123 cells cultured in the presence of methotrexate (MTX) at various concentration. The exposure of the cells to this drug resulted in a decrease of total and polymerised actin in cytoplasm and in some changes in actin filament organization. This coincided with a decrease of the cells' ability to migrate through Matrigel coated filters and with inhibition of tumour formation after reimplantation of the methotrexate treated cells to experimental rats.
Changes in the content of N-acetylneuraminic acid in rat erythrocyte membranes at different stages of experimental tumour (Morris hepatoma 5123) development were examined. Its content was lowered on the 30th and 40th day after transplantation of the tumour cells, as compared to the results for normal healthy rats. As a result of the tumour growth, the content of N-acetylgalactosamine, galactose and mannose in rat erythrocyte membranes became lowered, whereas that of glucose remained unchanged. The content of fucose was raised at early stage of tumour growth, and remained at this high level till the 40th day of experiment.
Fractions A (salted out by ammonium sulphate between 21 - 30% saturation), and fractions B (salted out between 51 - 70% saturation) of pyruvate kinase (EC 2.7.1.40.) corresponding respectively to pyruvate kinase types L and M2 from rat liver and Morris hepatoma 7777 were purified by an affinity chromatography on Blue Sepharose CL-6B. Peaks of inactive proteins were eliminated and the enzyme fractions bound biospecifically to the gels were eluted by free ADP. The molecular mass of purified hepatoma pyruvate kinase fraction B was smaller than that of liver pyruvate kinase fraction B. Morris hepatoma pyruvate kinase fraction B represented a variant of type M2, characterised by greatest affinity to 2-phosphoenolpyruvate as a main substrate and different sensitivity to low-molecular effectors in comparison with types L from both liver and hepatoma and in comparison with type M2 from normal rat liver. Only this hepatoma fraction B showed a tumour specific sensitivity to L-cysteine and was insensitive to normal signal molecules i.e. to ATP and fructose-l,6-diphosphate which influence liver pyruvate kinase activity. L-Cysteine inhibited the tumour fraction B of pyruvate kinase by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate.
In erythrocytes of rats bearing Morris hepatoma 5123 the activities of superoxide dismutase, glutathione peroxidase and glutathione reductase as well as the level of reduced glutathione increased on the 10th day after transplantation of the tumor. In the second phase of the tumor growth (20 days after transplantation), the activities of glutathione peroxidase, glutathione reductase and the level of reduced glutathione in erythrocytes of the experimental animals were lower than in controls, whereas the activity of superoxide dismutase was at that time higher than in controls. On the other hand, the activity of catalase did not significantly differ from that found in healthy rats.
Wistar and Buffalo rats of both sexes, aged 4 months, were divided into three groups: I which was given an intramuscular injection of 3 x 10 6 cells of Morris hepatoma (Buffalo males), II - subcutaneous injection of 3 x 10 4 cells of mammary gland carcinoma (Wistar females), III - intraperitoneal injection of 3 x 104 cells of Yoshid sarcoma (Wistar males). The animals were killed: in group I - 19, group II - 13 and in group III - 6 days after tumor transplantation. Twenty four hours before euthanasia the rats were given 5-brome-2'-deoxyuridine (BRd-U) at a dose of 50 mg/kg body mass. The control group consisted of animals with tumour. They were not treated with BRd-U. Im- munocytochemical reaction was performed on the sections of tumors, using monoclonal anti-BRd-U clone BU-33, Sigma. Computer measurements of tumor cells were carried out. There was a high similarity in morphological parameters between two kinds of cancer, and clear differences between them and Yoshid sarcoma. The main difference was noted in a twofold increase in the quantity of synthesised DNA in the nuclei of sarcoma cells. Immunocytochemical identification of tumor cells in phase S of the cell cycle with the use of monoclonal anti-BRd-U antibody is a precise and quick method of estimation of their proliferative potential.
The level of lipid peroxidation products and the content of glutathione in erythrocytes of rats with Morris 5123 hepatoma at different stages of tumor development were examined. The content of endogenous malondialdehyde (MDA) was increased throughout all periods of tumor development as compared to the results for healthy rats. From the extent of MDA generation under oxidative stress we concluded that erythrocytes of Morris 5123 hepatoma bearing rats were more susceptible to autoxidation than those from control rats. The content of reduced glutathione (GSH) and oxidized glutathione (GSSG) was increased at the early stage of tumor growth. At the advanced stage of the disease both the content of GSH and the GSH/GSSG ratio were decreased while the content of GSSG remained at the elevated level.
The highest amount of N-acetylneuraminic acid (AcNeu) was found in pyruvate kinase isoenzyme L from normal rat liver (24 moles/mole of enzyme tetramer), with the highest electrophoretic mobility. On the other hand, isoenzyme M2 from Morris hepatoma 7777, with the lowest electrophoretic mobility, had the lowest AcNeu content (5 moles/mole of enzyme tetramer). This tumour isoenzyme M2 of pyruvate kinase was, however, characterised by the highest phosphate content (12 moles/mole protein), in comparison to isoenzyme L (3 moles/mole protein) or normal liver isoenzyme M2 (6 moles/mole protein). This could indicate a regulatory change caused by reversible enzyme phosphorylation and dephosphorylation or sialization and desialization. Despite these differences, the sum of the two negatively charged residues was lower in tumour pyruvate kinase isoenzyme M2, with the slowest migration rate, than in normal rat liver isoenzyme M2. Moreover, isoenzyme M2 from tumour material, in comparison with isoenzyme M2 from normal rat liver, had a twice as high content of thiol groups (20 moles/mole protein), especially of free and superficially located ones, than the isoenzyme M2 from normal liver (10 moles/mole protein). This may explain abnormal susceptibility of tumour isoenzyme M2 to stereospecific inhibition by exogenous L-cysteine, and indicate genetically dependent changes in amino-acid content of tumour enzyme which take place during cell tumourigenic transformation.
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