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ePaper created 2017-01-04, 09:21:41 | version 1.38.0
| research 14 laser 4 2016 Calceinisabletopenetratethemembraneandisonly converted to a fluorescent colouring agent inside of an intact cell. If the cell membrane becomes perme- ableasaresultofdamages,calceinwillnotremainin- side the cell. As a consequence, Ethidium-homodi- mer-D1willenterthecellinexchange.Thissubstance is not permeable for intact membranes and will fluo- resce red when combined with DNA. Interestingly significant thermally-induced dam- ages were only observed at temperatures ranging from46.5 °C±0.5 °C.Startingatthistemperature,cell membranes are destroyed apparently. Temperatures from56.5 °C±0.5 °Cformanotherthresholdatwhich the 50 % lethality limit was reached. Ifthevitalitytestwasconducted24 hafterthermal treatment, almost twice as much lethal cells as ob- served1hafterincubationwereseenattemperatures from 46.5 °C to 56.5 °C. It appears that repairing pro- cesses cannot eliminate the thermal damage. Con- trarily, thermal treatment will result in a lethal reac- tion even 1 h later. Starting at 56.5 °C, most cells died immediately, probablyduetodenaturationoftheproteins(coagu- lation). Usually, a temperature level of 62 °C is given as the starting point for coagulation in the literature. However, the Life/Dead Assay does not allow any conclusionsontheeffectsofthedamagesonthecell organells, compartments or physiological reactions such as protein production. Consequently, HSP tests and electron microscopic examinations of the ultra- structure were conducted additionally. Heat-Shock-Proteins(HSP)weredetectedverywell at 50 °C by an antibody reaction. The cells were dis- tinctly coloured, which implies a significant reaction of the cell on the temperature-related stress. These cells were still able to synthesise the proteins and to survive for some time. Controls only showed only a light colouring, which may be the result of an un- specific reaction of the antibody with different cell proteins as well as a production of HSP which is not related to thermal stress. Similarly, a temperature level of 60 °C only lead to lightcolouration,whichcanbeexplainedbytheimme- diate lethal effect resulting in a missing time scale for the biosynthesis of HSP. In general, it should be noted thatthefirstHSPexaminationsdidnotexhibittheex- pectedintracellularresolutionduetoalowspecificity. The results of REM and TEM at the different guide values of 37 °C, 46.5 °C, 50 °C, 60 °C and 65 °C fit very wellwiththeresultsfromlightmicroscopy.Theeffects of a sudden and massive heating to more than 46 °C ontheexteriorcellshape(roundingandpartialreduc- tion of external structures) are distinctly visible. The extremelyfastcontractionofthecellsattemperatures around 50 °C might result in the observed tearing of cytoplasm-processes. Thermally-related membrane openingswerenotdetectedviaREMevenattempera- tures of 60 °C and above. These high temperatures probably resulted in an immediate coagulation of membrane proteins and other intracellular proteins, which lead to a “conservation” or fixation of the cells in their current shape. While the external cell shape was maintained because of the lacking time window for morphological modification, irreversible damages Fig. 8 Fig. 7 Fig. 7: TEM: Due to thermal treat- ment at 50 °C, the cells are rounded and the cell membrane forms vesicles (left). Mitochondria exhibit a disrupted structure of the christae, while the nuclear plasma starts to condense and the nucleus (K) itself often appears to be uncharacteris- tically flapped (arrow). The nuclear membrane (*) seems to be partially inflated or dissolved. Fig. 8: TEM: Thermal treatment at 60 °C. Parts of the cytoplasm are damaged or dissolved as can be seen by the mitochondria (M) with inflated or wound-up christae (arrows). The nucleus (K) shows severely condensed areas (*). 42016