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W Köhnlein

Publications and source records attributed to W Köhnlein.

At least 19 recordsLinked to original sources

Dosimetry of soft x-rays in thin liquid layers.

Very thin material layers (<100 microm) partially absorb ionizing radiation of low energy. When irradiating monolayer cell cultures from above, attention must be paid to absorption by the medium. Frequently, the volume of the nutrient medium is variable, and this leads to differences in the radiation doses delivered to the cells. In the present work these conditions were investigated for x-rays of energies between 13 kV and 100 kV in comparison with 60Co gamma rays using chemical dosimetry to measure the absorption by liquid layers between 25 microm and 500 microm thick. When the dose as measured with the ionization chamber was held constant, the dose absorbed in the Fricke solution was shown to increase with decreasing thickness of the layer of liquid because of a dose gradient. The effect of the dose gradient disappeared, however, in thick liquid layers of the Fricke solution by mixing during spectrophotometry. Secondary (photoeffect and Compton) electrons produced in air or filters are responsible for this effect in plastic petri dishes where back scattering at the interface does not occur. This interpretation is suggested by the same results of an analogous experimental setup using gamma rays with a 5-mm-thick Perspex plate. This dose increase in very thin layers, however, could not be verified by irradiating monolayer cells in poured-out plastic petri dishes because the secondary electrons are already absorbed in the remaining liquid film above the cells.

Animals↗

Mutagenic effect of low energy neutrons on human chromosome 11.

PURPOSE: The shape of the dose-effect curve for neutrons, i.e. the question as to whether the curve is linear or supralinear in the low-dose region, is still not clear. Therefore, the mutagenic effect of very low doses of low-energy neutrons was determined. MATERIALS AND METHODS: Human-hamster hybrid A(L) cells contain human chromosome 11, which expresses the membrane protein CD59. This membrane protein can be detected immunologically and quantified by flow cytometry. The A(L) cells were irradiated with neutrons of 0.565, 2.5 or 14.8 MeV and the results were compared with those after 200 kVp X-rays. Before irradiation, cells spontaneously mutated in the CD59 gene were removed by magnetic cell sorting (MACS). RESULTS: The relative biological effectiveness (RBE) for CD59 mutation induction was 19.8 (+/-2.7) for 0.565 MeV, 10.2 (+/-1.9) for 2.5 MeV, and 10.2 (+/-1.6) for 14.8 MeV neutrons. Linear mutation responses were obtained with all radiations except for 14.8 MeV neutrons where a supralinear curve may be a better fit. The deletion spectrum of mutated cell clones showed 29 Mbp deletions on average after irradiation with 0.069 Gy of 0.565 MeV neutrons. This scale of deletions is similar to that after 3 Gy 100 kV X-rays (=34 Mbp). For 50% cell survival, the RBE of the neutrons was 11 compared with 200 kV X-rays. CONCLUSIONS: Neutrons of low energies (0.565 or 2.5 MeV) produce a linear dose-response for mutation in the tested dose range of 0.015-0.15 Gy. The neutron curve of 14.8 MeV can be approximated by a curvilinear or linear function.

Animals↗

Identification of a deletion hotspot on distal mouse chromosome 4 by YAC fingerprinting.

Using repetitive elements as probes, genomic DNA fingerprints of four randomly selected yeast artificial chromosome (YAC) clones (two human and two mouse-derived YAC) were analyzed to determine the mutation level following X-ray exposure. Because the repetitive probes were derived from the mammalian host DNA, most of the fingerprint bands originated from the artificial chromosomes and not from the yeast genome. For none of the YAC clones was the mutation frequency elevated following X-ray exposure. However, for one mouse-derived YAC, the mutation level was unusually high (7%; 42 mutants of 607 clones analyzed), whereas for the other three YACs, the mutation level was nearly 0%. Surprisingly, 40 of the 42 mutations were deletions occurring only at three of the 20 mouse specific fingerprint bands. One of the frequently deleted fragments was cloned, sequenced and mapped to distal mouse chromosome 4, which has been repeatedly reported to be the most unstable region of the whole mouse genome, associated with various tumors. Deletion mapping of six YAC mutants revealed this fragment to be completely deleted in four YACs. In the other two mutants, recombination occurred within the fragment, in each case initiated at the same LINE-1 element. In conclusion, the presented YAC fingerprint is a useful tool for detecting and characterizing unstable regions in mammalian genomes.

Animals↗

Frequency of CD59 mutations induced in human-hamster hybrid A(L) cells by low-dose X-irradiation.

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.

Animals↗

Pulsed-field gel electrophoresis of chromosomal DNA of Saccharomyces pastorianus after exposure to x-rays (30-50 keV) and neutrons (14 MeV).

Chromosomes of budding yeast Saccharomyces pastorianus were used to determine the extent of DNA double-strand breaks (DSBs) induced by x-rays (30-50 keV) and 14 MeV neutrons. The yeast chromosomes were separated by pulsed-field gel electrophoresis (PFGE) and the proportion of unbroken molecules corresponding to the largest chromosome no. IV (1500 kbp) was used to calculate the DSB frequency assuming a random distribution of hits. To determine the protective contribution of the cell environment, chromosomes embedded in agarose plugs as well as intact yeast cells, were irradiated under conditions completely inhibiting DNA repair. Following irradiation, the intact cells were also embedded in agarose plugs and the chromosomes isolated to perform PFGE. All radiation experiments resulted in a linear dose-effect curve for DSBs. For both radiation qualities, the yield of DSBs for exposed isolated chromosomes exceeded that for intact yeast cells by a factor of 13. The relative biological effectiveness (RBE) of 14 MeV neutrons in the induction of DNA DSBs was about 2.5. This figure was found to be identical for the in vivo and in vitro exposure of yeast chromosomes (neutrons 36.7 and 2.8, x-rays 14.5 and 1.1 x 10(-8) DSB x Bp-1 Gy-1 for isolated DNA and intact cells, respectively).

Chromosomes, Fungal↗

The relative biological effectiveness of low doses of 14 MeV neutrons in steady-state murine spermatogenesis as determined by flow cytometry.

The relative biological effectiveness of 14 MeV neutrons in the low-dose range < or =1 Gy has been determined in differentiating and differentiated spermatogonia. Male NMRI mice were exposed to single doses of 2 cGy to 3 Gy of (60)Co gamma rays or neutrons. The ratios of testicular S-phase cells, 4c primary spermatocytes, and elongated spermatids were quantified by DNA flow cytometry 2 to 70 days after irradiation and were found to decrease. Histological samples and testis weight were analyzed in parallel. Doses of 2-5 cGy neutrons and 10-50 cGy gamma rays significantly (P<0.05) decreased the proportions of S-phase cells, spermatocytes and elongated spermatids at 4, 14 and 28 days postirradiation. For S-phase cells, the biphasic shape of the cell survival curves was described with a D(50) of 5 cGy neutrons. The D(50) for (60)Co gamma rays and the relative biological effectiveness could not be determined. The relative biological effectiveness of neutrons at 50% reductions of testis weight, primary spermatocytes, and elongated spermatids were 2.5, 10.0 and 6.1, respectively. This in vivo assay is interesting because of its sensitivity at dose ranges that are relevant for exposures in the environment, the workplace and radiotherapy.

Animals↗

Keratinocytes exposed to ultraviolet radiation reveal three down-regulated genes with potential function in differentiation and cell cycle control.

The incidence of skin cancer is increasing in epidemic proportion. Although solar UV radiation is known to be the major risk factor, much information is lacking about the molecular mechanisms leading to skin cancer. To gain a deeper insight into these mechanisms, we have examined cells of a human keratinocyte cell line (HaCat) after exposure to 0.16 minimal erythema doses of UVB radiation. This dose led to an S-phase delay that was reversible 22 h postirradiation. To examine gene expression 10 h after UV irradiation, a nonradioactive differential display was employed. Three genes were identified as being down-regulated significantly. The first encodes for topoisomerase-IIbeta-binding protein 1 (expression level 5% 6 h after irradiation). This protein is associated with human topoisomerase IIbeta and appears to be necessary for DNA replication during the onset of S phase. The second gene product has previously been reported to be involved in differentiation and is therefore known as differentiation-dependent A4 protein (28% 8 h after irradiation). The third gene is XPO1 (also known as CRM1) (5% 8 h after irradiation), whose protein is involved in nuclear export of mRNA molecules. Differential expression of these genes after UV irradiation has not been reported. Because of their potential involvement in cell cycle control and differentiation, these proteins could be important for understanding the reaction of keratinocytes after exposure to UV radiation.

Carrier Proteins↗

Effects of single and split doses of cobalt-60 gamma rays and 14 MeV neutrons on mouse stem cell spermatogonia.

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy (60)Co gamma rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy (60)Co gamma rays or 3 Gy neutrons and observed 70 days later. Split doses of (60)Co gamma rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.

Animals↗

Nonisotopic detection of mutations using a modified single-strand conformation polymorphism analysis.

This report describes a rapid convenient screening system with improved sensitivity to detect mutations in the cystic fibrosis transmembrane regulator (CFTR) gene based on nonisotopic SSCP analysis. Because conventional SSCP analysis is often hampered by poor yield of single-stranded DNA, we applied the well-established solid-phase technique in which streptavidin-coated magnetic beads are used to immobilize biotinylated polymerase chain reaction (PCR) products. High yield of single-stranded DNA can be eluted from the solid phase by denaturation and used for SSCP analysis. An additional advantage of this procedure is that the immobilized single strand is available without any further purification steps for solid-phase sequencing.

Base Sequence↗

Inconsistencies and open questions regarding low-dose health effects of ionizing radiation.

The effects on human health of exposures to ionizing radiation at low doses have long been the subject of dispute. In this paper we focus on open questions regarding the health effects of low-dose exposures that require further investigations. Seemingly contradictory findings of radiation health effects have been reported for the same exposed populations, or inconsistent estimates of radiation risks were found when different populations and exposure conditions were compared. Such discrepancies may be indicative of differences in sensitivities among the applied methods of epidemiological analysis or indicative of significant discrepancies in health consequences after comparable total exposures of different populations under varying conditions. We focus first on inconsistencies and contradictions in presentations of the state of knowledge by different authoritative experts. We then review studies that found positive associations between exposure and risks in dose ranges where traditional notions (generalized primarily from high-dose studies of A-bomb survivors or exposed animals) would have predicted negligible effects. One persistent notion in many reviews of low-dose effects is the hypothesis of reduced biological effectiveness of fractionated low-dose exposures, compared to that of the same acute dose. This assumption is not supported by data on human populations. From studies of populations that live in contaminated areas, more and more evidence is accumulating on unusual rates of various diseases other than radiation-induced malignancies, health effects that are suspected to be associated with relatively low levels of internal exposures originating from radioactive fallout. Such effects include congenital defects, neonatal mortality, stillbirths, and possibly genetically transmitted disease. A range of open questions challenges scientists to test imaginative hypotheses about induction of disease by radiation with novel research strategies.

Animals↗

The acidic groups of the neocarzinostatin protein play an important role in its biological activity.

Despite the extensive literature on the mechanism of action of the anticancer agent neocarzinostatin (NCS), the role of the protein moiety is still not clear. The model involving endocytosis of intact holo-NCS has been dismissed in favor of a theory proposing entry of free dissociated chromophore. However, the fact that the NCS protein has a certain affinity for cell membranes cannot be disregarded. In the present work the protein moiety has been modified by transformation of the carboxyl groups into isopropylamide groups. This modification resulted in a broad shift of the protein pI towards higher values, accompanied by a marked reduction of toxicity in vitro and reduced binding of protein to cells. Coincubation of modified NCS with inactive native apo-NCS led to restoration of the biological activity of native holo-NCS. It has been shown chromatographically that the modified NCS is capable of transferring active chromophore to native apo-NCS. The inactive form (apoprotein) of the charge-modified NCS, however, is capable of inhibiting the toxicity of the active form, as has been described for the corresponding native pair. Binding experiments with tritium-labeled proteins revealed that the modified protein has diminished affinity for membranes, in comparison with native NCS.

Agar↗

Free radicals from irradiated lyophilized DNA: influence of water of hydration.

Lyophilized DNA equilibrated with water vapour at various relative humidities (0-95% H2O or D2O) was X-irradiated at 77 K and analysed for free radicals by electron paramagnetic resonance (EPR) spectroscopy in the temperature range 77-280 K. Analysis of spectra according to variation in humidity, microwave power and temperature generally yielded a doublet and a triplet spectrum at 77 K. The doublet partially converted into the 5-thymyl radical (TH.). DNA containing deuterated thymine (dTDNA) revealed that the doublet of 'normal' DNA should be composed of two similar doublets, one of which should be assigned to the thymine anion, the other possibly the cytosine anion. The triplet signal was more stable and could be related to the guanine cation or its deprotonated successor. Several other patterns were detected, among them an allyl radical in highly aquated DNA (95% humidity). Other features occurred either predominantly or exclusively in DNA equilibrated above 66% relative humidity and were ascribed to an influence of the secondary structure. Among them were components possibly indicating H- or D-addition to the cytosine base, a reaction also derivable for dTDNA. Quantitative analysis of the total radical yield and the relative TH. concentration revealed that one cause of the dominance of the latter is its thermal stability vs. other species. The total radical concentration increases with target size up to 76% relative humidity, the hydration water forming an integral part of the DNA. At 95% relative humidity OH. radicals are formed in addition to DNA radicals, showing that ice and hydrated DNA have separated into discrete targets.

DNA↗

Strand breaks in plasmid DNA, natural and brominated, by low-energy X-rays.

Single- and double-strand breaks were measured in Col E1 plasmid DNA, natural and partially brominated, irradiated with monoenergetic X-rays (from a synchrotron radiation source) on both sides of the K-absorption edge for Br. The fraction of the undamaged supercoiled form decreased exponentially with the photon dose; its yield in the brominated DNA did not exhibit any energy-dependence. This result is consistent with the calculated relative contributions of photoelectric interactions with individual component atoms which show that an electron flux originating in light atoms outweighs that from bromine. However, X-rays of energy above the Br K-edge appear slightly more efficient in producing double-strand breaks. This result, as well as those reported in the literature on the Auger enhancement of different biological endpoints in cells containing brominated DNA, seem to suggest that the positive charge and submolecular effects associated with the photo-absorption in bromine play some role in damaging processes, besides the initial distribution of deposited electron energy.

Bromine↗

Increased serum stability and prolonged biological half-life of neocarzinostatin covalently bound to monoclonal antibodies.

The pharmacokinetics of neocarzinostatin (NCS) have been compared to NCS conjugates with monoclonal antibodies using Balb/c and tumor bearing nude mice. Data on blood and whole body clearance revealed that the high MW conjugate persists in the body far longer and at a higher level than the free drug. Excretion of the free drug occurs with an extremely rapid renal clearance and localization of the remaining drug in the kidney, whereas the NCS immunoconjugate remained in circulation far longer allowing time for tumor localization to occur without renal accumulation of drug. In addition, NCS conjugated to monoclonal antibody was found to retain its activity in human serum better than free drug, in agreement with data obtained for other NCS-derivatives. Half-time of inactivation was greatly extended when measured under relevant conditions in a DNA strand-break assay. The results indicate that two of the most important requirements for the successful targeting of NCS in vivo, decreased clearance rate and increased serum stability are achieved by conjugation to antibody. Both results increase the probability of NCS accumulating in tissue while still in its active form. Coupling of NCS to monoclonal antibody decreases clearance and inactivation rate and increases localization of the active drug in tumor tissue.

Animals↗