Modulation of signal transduction pathways and global protein composition of macrophages by ionizing radiation.

It is assumed that the exposure of cells to ionizing radiation modulates their signal transduction pathways, which then govern the early and late radiation-induced alterations in gene expression. In this study we tested the effects of low doses of X-irradiation on the cell signaling and global protein composition of an HL-60 human promyelocytic leukemia cell line differentiated along a macrophage-like cell pathway by 4beta-phorbol-12-myristate-13-acetate (PMA). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting of anti-phosphotyrosine immunoprecipitates, we found radiation-induced changes in the level of phosphorylation of proteins with molecular masses of 45 and 48 kDa, but in the most intensively stained area, ranging from 54 to 60 kDa, no alterations were observed. When two-dimensional electrophoresis (2-DE) immunoblotting was applied, only proteins from this heavily stained region were visualized and in addition the evident differences in tyrosine phosphorylated protein patterns between nonirradiated and irradiated cells were found in this area. Furthermore, the immunostaining of extracellular signal-regulated kinase 2 (ERK2) which did not prove its tyrosine phosphorylation demonstrated the existence of several ERK2 charge isoforms showing differential expression after X-irradiation. Comparing the whole protein profiles we found after the simultaneous quantitation of 1000 matched spots two proteins whose expression was regulated in an opposite manner in nonirradiated and X-irradiated cells. The quantities of both spots showed increases or decreases by a factor of 2 or more between irradiated and nonirradiated samples and both these changes were statistically significant (P<0.05).

Isolation of micronuclei: high yield by sucrose gradient versus maximum purity by cell sorting.

Micronuclei (MNC) of L929 cells were isolated 72 h after irradiation with 6 Gy for characterization of their DNA content, using gel electrophoresis. A novel method for isolation of MNC based on a sucrose gradient ultracentrifugation was developed. With this efficient method (80% recovery) more than 150 x 10(6) MNC per day could be isolated. The purity was > 99%. However, the low number of main nuclei in the MNC isolate ( < 1%) resulted in a contamination of MNC DNA with about 15% main nucleus DNA, due to the several times higher DNA content of main nuclei. Cell sorting was utilized to maximize the purity by choosing the recommended sorting mode for highest purity. Isolation of MNC with the cell sorter was successful (100% purity), but also time-consuming (1-2 x 10(6) MNC per working day could be isolated) and insufficient (10% recovery). Extraction of DNA of these isolated MNC resulted in less than 1 ng/day. Hence, at least 1 week of cell sorting would be necessary for one electrophoretic run. When employing the sucrose gradient method, 2000 times more DNA of MNC have been isolated. We therefore consider this method as the most efficient way for rapid and low cost isolation of large amounts of purified ( > 99%) MNC without the employment of sophisticated and expensive techniques (cell sorting) and the accompanied knowhow. In contrast, maximum purity but low yields of MNC can be obtained by cell sorting.

[Prognostic value of electrodiagnosis of Bell's palsy]. / Zur prognostischen Wertigkeit der Elektrodiagnostik bei der Bell'schen Lähmung.

Many papers report on a poor rate of complete restitution of Bell's palsy if signs of degeneration can be detected in neuromyography (NMG) or electromyography (EMG). In 119 patients who underwent infusion therapy (as developed by Stennert) 39% showed signs of degeneration in EMG or NMG. Complete restitution was achieved in 93% of these patients. Degeneration was more frequent in elderly patients (< 20 years: 20%, > 60 years: 55%). This did not affect the rate of complete restitution, which was constantly high for every age. If infusion therapy was started within 7 days after onset of the disease, no defects in restitution were observed, which was frequently so if therapy was started later. After one year the rate of complete restitution was about equal in cases with signs of degeneration (91%) and non-degenerative cases (94%). But 80% of the non-degenerative cases showed complete restitution within 3 months after onset of the palsy, whereas 80% of cases with signs of degeneration healed after this date (mean 6.1 months). After oral therapy with cortisol exclusively half of the degenerative cases did not attain complete restitution. After infusion therapy EMG and NMG do not answer the question if a Bell's palsy will heal completely or not but enable us to predict when this will probably be the case.

Cell kinetic analysis after irradiation in L929- and LLC-MK2-cells by a BrdU/DNA assay.

Exponentially growing L929- and LLC-MK2-cells were X-irradiated in vitro. Irradiation-induced cell cycle kinetic effects were examined by the calculation of the cell number doubling time (TD), the duration of the cell cycle phases by the BrdU/DNA assay and the number of micronuclei. The number of cells arrested in G2/M and the duration of the delay are dose-related. The effect of irradiation on the duration of cell cycle phases was estimated by following the BrdU-labeled cells through the cell cycle. With increasing radiation doses the duration of the G2/M-phase increases whereas G1- and S-phase show only slight variations. Due to the problems involved in evaluation of radiation effects on the cell cycle a comparison with other methods proves the BrdU/DNA-assay to be a valuable instrument in those experiments. The micronucleus frequency is dependent on irradiation dose. However, after 7.52 Gy the number of micronuclei increases, whereas no cells in G1-phase and no increase of cell number could be detected, indicating a mechanism of micronucleus formation that is not linked with mitosis.