Biomedical subjects
Publications and source records attributed to B Konze-Thomas.
The DFG is the major funding organisation for research at German universities. A considerable amount of funds are spent on clinical research, including surgical research. Despite some success, the quality of clinical research in Germany is not considered to be satisfactory. Where are the deficits, and how is it possible to improve the situation? By which mechanisms could the DFG add to the improvement of clinical research?
The Deutsche Forschungsgemeinschaft (DFG) serves all fields of sciences and the arts by financing research projects. The DFG can support science by funding of individual grants, priority programmes, research groups and collaborative research centers. With these mechanisms of funding, clinical research is also supported. Some structural aspects of clinical research and a few thoughts for the improvement of clinical research are presented.
Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G0 state. Autoradiography studies of cells released from G0 after 72 h and replated at lower densities (3-9 x 10(3) cells/cm2) in fresh medium containing 15% fetal bovine serum showed that semi-conservative DNA synthesis (S phage) began approximately 24 h after the replating. To determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts, we released cultures of NF or XP12BE cells from G0, allowed them to reattach at lower densities, irradiated them in early G1 (approximately 18 h prior to the onset of S) or just prior to S phase, and assayed the frequency of mutations to 6-thioguanine resistance and the survival of colony-forming ability. The XP12BE cells, which are virtually incapable of excising UV-induced DNA lesions, showed approximately the same frequency of mutations and survival regardless of the time of UV irradiation. In NF cells, the slope of the dose response for mutations induced in cells irradiated just prior to S was about 7-fold steeper than that of cells irradiated 18 h earlier. However, the two sets of NF cells showed no significant difference in survival. Neither were there significant differences in the survival of NF cells released from G0, plated at cloning densities and irradiated as soon as they had attached and flattened out. (approximately 20 h prior to S) or 4, 8, 12, 16, 20, or 24 h later. We conclude that the frequency of mutations induced by UV is dependent upon the number of unexcised lesions remaining at the time of semi-conservative DNA replication. However, the amount of time available for excision of potentially cytotoxic lesions is not determined primarily by the period between irradiation and the onset of S phase.
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We have shown that the replication of avian sarcoma/leukemia oncornaviruses is not entirely dependent on the metabolic functions of the host cell. It is demonstrated that the proteolytic enzyme which processes the precursor protein pr76 to the group specific antigen (gag) proteins, is virally coded and hence functions independently of cellular activities. It is the gag protein p15 and it cleaves its own precursor. This protease is also able to cleave in vitro the polyprotein of 110 kd which is synthesized in cells non-productively infected with the defective leukemia virus MC29 but not cleaved.
The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.
The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (a) removal of thymine-containing dimers, (b) DNA excision repair synthesis, and (c) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis.
RNA polymerase was isolated from Escherichia coli and Serratia marcescens. The subunits of both enzymes were separated by electrophoresis on cellulose acetate sheets in the presence of urea. Under conditions favouring reconstitution of the RNA polymerases, stoichiometric amounts of the subunits were allowed to interact. Active hybrid enzymes were formed if corresponding subunits of both enzymes were mutually exchanged. The analysis of the RNA products synthesized showed that the reconstituted enzymes are able to recognize the promoters for transcription and the termination signals on the DNA template. The transcription products can serve as messengers for cell-free protein synthesis.