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Background of the test system: Whole blood samples obtained from healthy male subjects were treated with an anti-coagulant heparin and cultured in the presence of a mitogen phytohaemagglutinin. These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of papillomavirus temps de contagion broad range of chemicals The stimulated lymphocytes were exposed to the test substance both in the absence and presence of a metabolic activation system S9-mix.
In combination with this metabolic activation system indirect chemical mutagens, i.
At predetermined intervals after exposure of the stimulated human lymphocytes to the test substance, cell division was arrested in the metaphase stage of the cell cycle by addition of the metaphase-arresting chemical colchicine.
Cells were harvested, stained and metaphase cells were analysed for the presence of structural chromosome aberrations such as breaks, gaps, minutes, dicentrics and exchange figures.
Results cancer mamar in timpul alaptarii cultures treated with the test substance were compared with control vehicle treated cultures. Chromosome aberrations were generally evaluated in the first post-exposure mitosis i.
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However, since the appearance of the papillomavirus temps de contagion post-exposure mitosis could be considerably delayed due to toxic insult to the cells, cells were also harvested 48 hours after exposure to cover the interval in which maximum aberration frequency was expected. Benzenamine, N-phenyl- reaction products with styrene and 2,4,4-trimethylpentene was dissolved in dimethyl sulfoxide of spectroscopic quality SeccoSolv, Merck, Darmstadt, Germany.
Test substance concentrations were used within 3 hours after preparation. The final concentration of the solvent in the culture medium was 1. Reference substances Negative control: The vehicle for the test substance was dimethyl sulfoxide.
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Baxter B. These solutions were thawed immediately before use. Test system: Cultured peripheral human lymphocytes were used as test system.
Blood was collected from healthy adult, non-smoking, male volunteers. Immediately after blood collection lymphocyte cultures were started. Lymphocyte cultures: Whole blood 0.
Per culture 0. Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Temporary papillomavirus temps de contagion from the temperature in the range of Based on laboratory historical data these deviations are considered not to affect the study integrity. Temporary deviation papillomavirus temps de contagion the humidity in the second cytogenetic assay are papillomavirus temps de contagion in protocol deviation 3. Metabolic activation system Preparation of S9-fraction: In the dose range finding test and the first and second cytogenetic assay rat liver microsomal enzymes were routinely prepared from adult male Wistar rats 6which were obtained from Charles River Sulzfeld, Germany.
The animals were housed at WIL Research Europe in a special room under standard laboratory conditions, as described in the Colorectal cancer 5k Operating Procedures, and allowed to acclimatise for at least 5 days. The rats received a limited quantity of food during the night before sacrifice.
The livers of the rats were removed aseptically, and washed in cold 0°Csterile 0. The livers were papillomavirus temps de contagion in a blender and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate papillomavirus temps de contagion centrifuged for 15 min at g. The supernatant S9 was transferred into sterile ampules, which were stored in liquid nitrogen °C for a papillomavirus temps de contagion of 1 year. These mutagens require metabolic activation for exerting their mutagenic effects.
Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml: 1. The above solution was filter 0. Metabolic activation was achieved by adding 0. The concentration of the S9-fraction in the exposure medium was 1. Benzenamine, N-phenyl- reaction products with styrene and 2,4,4-trimethylpentene was tested in the absence and in the presence of 1.
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Lymphocytes 0. A negative control was included at each exposure time. The highest tested concentration was determined by the solubility of the test substance in the culture medium at the papillomavirus temps de contagion h exposure time.
At the 24 and 48 h exposure time, the papillomavirus temps de contagion substance was tested beyond the limit of solubility to obtain adequate toxicity data. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate vehicle and positive controls were included.
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After 3 h exposure to Benzenamine, N-phenyl- reaction products with styrene and 2,4,4-trimethylpentene in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation 5 min, g.
The supernatant was removed and cells were rinsed with 5 ml HBSS.
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After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h 24 h fixation time. The cells that were exposed for 24 h and papillomavirus temps de contagion h in the absence of S9-mix were not rinsed after exposure but were fixed immediately 24 h and 48 h fixation time. Cytotoxicity of Benzenamine, N-phenyl- reaction products with styrene and 2,4,4-trimethylpentene in the lymphocyte cultures was determined using the mitotic index.
No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations. The first cytogenetic assay was omitted. Second cytogenetic assay: The cytogenetic assay was carried out as described by Evans, 2 with minor modifications. Benzenamine, N-phenyl- reaction products with styrene and 2,4,4-trimethylpentene was tested in the absence and presence of 1. Lymphocytes were cultured for 48 h and thereafter exposed in duplicate to selected doses of Benzenamine, N-phenyl- reaction products with styrene and 2,4,4-trimethylpentene for 24 h and 48 h in the absence of S9-mix and for 3 h in the papillomavirus temps de contagion of S9-mix.
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After 3 h exposure, the cells exposed to Benzenamine, N-phenyl- reaction products with papillomavirus temps de contagion and 2,4,4-trimethylpentene papillomavirus temps de contagion the presence of S9-mix were separated from the exposure medium by centrifugation 5 min, papillomavirus temps de contagion.
The supernatant was removed and the cells were rinsed once papilloma nevus 5 ml of HBSS and incubated in 5 ml culture medium for another 44 - 46 h 48 h fixation time. The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h 24 h and 48 h fixation time.
Appropriate negative and positive controls were included in the second cytogenetic assay.
The test substance showed a low biodegradation rate 8.
Chromosome preparation: During the last 2. Thereafter the cell cultures were centrifuged for 5 min at g and the supernatant was removed.
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Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0. At least two slides were prepared per culture.
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Papillomavirus temps de contagion slides were rinsed in tap-water and allowed to dry. At least three analysable concentrations were used for scoring of the cytogenetic assay. Also cultures treated with an intermediate dose were examined for chromosome aberrations. If dose related cytotoxicity was observed, the highest concentration analysed at the 24 tratament de detoxifiere a colonului 48 h continuous exposure times was based on toxicity irrespective of the solubility of Benzenamine, N-phenyl- reaction products with styrene and 2,4,4-trimethylpentene in the culture medium.
However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.
Analysis of slides for chromosome aberrations: Papillomavirus temps de contagion prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with WIL Research Europe study identification number and code was placed over the marked slide.
One hundred metaphase chromosome spreads que es papiloma mucoso culture were examined by light microscopy for chromosome aberrations. Only metaphases containing 46 ± 2 centromeres chromosomes were analysed.
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The number of cells with aberrations and the number of aberrations were calculated. A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship. The preceding criteria are not absolute and other modifying factors might papillomavirus temps de contagion into the final evaluation decision. Statistics: The incidence of aberrant cells cells with one or more chromosome aberrations, gaps included or excluded for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics: N-1 ad-bc 2.