Biomedical subjects

T Bauch

Publications and source records attributed to T Bauch.

15 recordsLinked to original sources

PubMed2005

Collapse of thermal activation in moderately damped Josephson junctions.

We study switching current statistics in moderately damped Nb-InAs-Nb and intrinsic Bi2Sr2CaCu2O8+delta) Josephson junctions. A paradoxical collapse of thermal activation with increasing temperature is reported and explained by the interplay of two conflicting consequences of thermal fluctuations, which can both assist in premature escape and help in retrapping back into the stationary state. We analyze the influence of dissipation on the thermal escape by tuning damping with a gate voltage, magnetic field, temperature, and an in situ capacitor.

Journal Article↗

PubMed2005

Macroscopic quantum tunneling in d-wave YBa2Cu3O7-delta Josephson junctions.

The escape rate from the zero voltage state in a superconducting Josephson junction (JJ) is determined by the temperature, but it saturates at low temperature due to macroscopic quantum tunneling (MQT). Complications due to d-wave symmetry in a high temperature superconductor, like low energy quasiparticles and an unconventional current-phase relation, may influence the escape rate. We report, for the first time to our knowledge, the observation of MQT in a YBa(2)Cu(3)O(7-delta) grain boundary biepitaxial JJ. This proves that dissipation can be significantly reduced by a proper junction configuration, which is of significance for quantum coherence.

Journal Article↗

PubMed2002

BRCA1 and BRCA2 heterozygosity and repair of X-ray-induced DNA damage.

PURPOSE: Up to 90% of hereditary breast cancer cases are linked to germ-line mutations in one of the two copies of the BRCA1 or BRCA2 genes. Brca1 and Brca2 proteins are both involved in the cellular defence against DNA damage, although the precise function of the proteins is still not known. Some studies on a small number of samples as well as the present pilot study also suggested that BRCA1 heterozygosity may lead to impaired repair of ionizing-radiation-induced DNA double-strand breaks. The purpose of the study was to test in a larger family-matched study whether carriers of BRCA1 or BRCA2 mutations have an increased sensitivity to ionizing radiation. MATERIALS AND METHODS: In a blind study, the effect of different germ-line mutations in one allele of the BRCA1 or BRCA2 gene on the ability to repair X-ray-induced DNA breaks was investigated. Fibroblasts and lymphocytes were taken from heterozygotic individuals (BRCA1+ /- and BRCA2+ /-) with different mutations and from relatives proven to be non-carriers of the BRCA mutations. Rejoining of DNA breaks was analysed by pulsed-field gel electrophoresis (for fibroblasts) or the comet assay (for lymphocytes). RESULTS: Significant interindividual differences were found in the capacities of the fibroblasts and lymphocytes to rejoin DNA breaks induced by X-radiation. However, these differences were not related to heterozygosity in BRCA1 or BRCA2. CONCLUSIONS: Cells from carriers of mutations in one allele of the BRCA1 or BRCA2 genes have no gross defects in their ability to rejoin radiation-induced DNA breaks. Hence, these carriers may not be at risk of developing excess normal tissue reactions after radiotherapy consistent with data from recent clinical studies.

Breast Neoplasms↗

PubMed2002

Does radiotherapy affect the outcome of the comet assay?

This study was designed to assess possible effects of fractionated radiotherapy (5 or 10 fractions at 2 Gy per fraction) on the DNA repair capacity of lymphocytes, as measured by the comet assay. 50 patients with various tumour types were chosen. They had received no chemotherapy during the 6 months prior to radiotherapy and did not receive cortisone. 10 ml of heparinized blood was collected before radiotherapy, after 5 fractions and after 10 fractions. Lymphocytes were isolated and analysed using the comet assay. On average, no effect on DNA repair capacity was observed that could be attributed to radiotherapy. On an individual basis, there were a few patients who showed a comparatively pronounced variability in their response to radiotherapy (three patients with a relative coefficient of variability of more than 30%). There was some indication of a weak correlation between poor repair capacity and severe side effects in normal tissue. We also found that alcohol in particular, and smoking to some extent, may impair repair capacity during radiotherapy. Age, gender, field size, medication and tumour entity showed no effect on repair capacity.

Alcohol Drinking↗

PubMed2001

Heterogeneity in 2-deoxy-D-glucose-induced modifications in energetics and radiation responses of human tumor cell lines.

PURPOSE: The glucose analog and glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), has been shown to differentially enhance the radiation damage in tumor cells by inhibiting the postirradiation repair processes. The present study was undertaken to examine the relationship between 2-DG-induced modification of energy metabolism and cellular radioresponses and to identify the most relevant parameter(s) for predicting the tumor response to the combined treatment of radiation + 2-DG. METHODS AND MATERIALS: Six human tumor cell lines (glioma: BMG-1 and U-87, squamous cell carcinoma: 4451 and 4197, and melanoma: MeWo and Be-11) were investigated. Cells were exposed to 2 Gy of Co-60 gamma-rays or 250 kVP X-rays and maintained under liquid-holding conditions 2-4 h to facilitate repair. 2-DG (5 mM, equimolar with glucose) that was added at the time of irradiation was present during the liquid holding. Glucose utilization, lactate production (enzymatic assays), and adenine nucleotides (high performance liquid chromatography and capillary isotachophoresis) were investigated as parameters of energy metabolism. Induction and repair of DNA damage (comet assay), cytogenetic damage (micronuclei formation), and cell death (macrocolony assay) were analyzed as parameters of radiation response. RESULTS: The glucose consumption and lactate production of glioma cell lines (BMG-1 and U-87) were nearly 2-fold higher than the squamous carcinoma cell lines (4197 and 4451). The ATP content varied from 3.0 to 6.5 femto moles/cell among these lines, whereas the energy charge (0.86-0.90) did not show much variation. Presence of 2-DG inhibited the rate of glucose usage and glycolysis by 30-40% in glioma cell lines and by 15-20% in squamous carcinoma lines, while ATP levels reduced by nearly 40% in all the four cell lines. ATP:ADP ratios decreased to a greater extent ( approximately 40%) in glioma cells than in squamous carcinoma 4451 and MeWo cells; in contrast, presence of 2-DG reduced ADP:AMP ratios by 3-fold in the squamous carcinoma 4451, whereas an increase was noted in the glioma cell line BMG-1. 2-DG significantly reduced the initial rates of DNA repair in all cells, resulting in an excess residual damage after 2 h of repair in BMG-1, U-87, and 4451 cell lines, whereas no significant differences could be observed in the other cell lines. Recovery from potentially lethal damage was also significantly inhibited in BMG-1 cells. 2-DG increased the radiation-induced micronuclei formation in the melanoma line (MeWo) by nearly 60%, while a moderate (25-40%) increase was observed in the glioma cell lines (BMG-1 and U-87). Presence of 2-DG during liquid holding (4 h) enhanced the radiation-induced cell death by nearly 40% in both the glioma cell lines, while significant effects were not observed in others. CONCLUSIONS: The modifications in energetics and radiation responses by 2-DG vary considerably among different human tumor cell lines, and the relationships between energy metabolism and various radiobiologic parameters are complex in nature. The 2-DG-induced modification of radiation response does not strictly correlate with changes in the levels of ATP. However, a significant enhancement of the radiation damage by 2-DG was observed in cells with high rates of glucose usage and glycolysis, which appear to be the two most important factors determining the tumor response to the combined treatment of 2-DG + radiation therapy.

Adenosine Diphosphate↗

PubMed2001

Radiation sensitivity of lymphocytes from healthy individuals and cancer patients as measured by the comet assay.

Lymphocytes of healthy volunteers (n = 24) and of tumour patients (n = 30, 18 of whom had experienced severe side-effects) were irradiated with x-rays in vitro. DNA damage was analysed after 0.25-2 Gy and DNA repair after 2 Gy, and quantification of both endpoints was done by the comet assay. The individual differences in radiation-induced DNA damage as well as in the repair kinetics were observed to be striking for both healthy donors and tumour patients. After a repair time of 3 h, following 2 Gy x-irradiation, some of the healthy volunteers showed no residual DNA damage at all in their lymphocytes, whereas others revealed about 30%. There was no indication that our results were affected by either age, gender or smoking habits. Slow repair kinetics and high amounts of residual damage were characteristic for many but not all tumour patients who had experienced severe side-effects in their normal tissues during or after radiotherapy (n = 18). Our conclusion is that those individuals showing poor DNA repair characteristics in the lymphocytes following in vitro irradiation, have a high probability of being radiosensitive. The opposite conclusion is not necessarily true: if repair is effective, this does not mean that the individual is radioresistant, because factors other than impaired repair may cause radiosensitivity.

Comet Assay↗

PubMed2000

DNA excision repair and DNA damage-induced apoptosis are linked to Poly(ADP-ribosyl)ation but have different requirements for p53.

Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD(+) to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death.

Animals↗

PubMed1999

Automated comet assay analysis.

BACKGROUND: Recently the "comet assay" or "single-cell gel electrophoresis assay" has been established as a sensitive method for the detection of DNA damage and repair. Most of the software now available to quantify various parameters for DNA damage requires the interaction of a human observer. In this report, we describe an automated analysis system that is based on self-developed software and hardware and needs minimal human interaction. METHODS: The image analysis is divided into two parts: 1) automatic cell recognition and comet classification and 2) quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (Böcker et al.: Phys Med Biol 1997;42:1981-1992) allows for faster analysis. RESULTS: Our recognition software shows a sensitivity of 95.2% and a specificity of 92.7% when tested on test samples from routine work with DNA damage by low-dose radiation (0-2 Gy). The parallel hardware and software concept enables us to analyze 100 comets on one slide in less than 15 min. CONCLUSIONS: A comparison of measurements made on the same samples by manual and automated analysis systems revealed that there are no significant differences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possible.

Algorithms↗

PubMed1999

Optimization and standardization of the "comet assay" for analyzing the repair of DNA damage in cells.

BACKGROUND: The "comet assay" has become an interesting and a very useful tool for the analysis of the induction and amount of DNA damage in single cells thus offering the opportunity to measure the effectiveness of DNA repair. On the basis of the Ostling and Johanson protocol we have developed a modified method with increased sensitivity and high reproducibility. MATERIAL AND METHODS: Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique. RESULTS: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for "comet" analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. CONCLUSIONS: This paper presents data on the optimization and standardization of the original "comet assay" leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the "comet assay" may have the potential for the estimation of individual radiosensitivity.

Adult↗

PubMed1997

Image analysis of comet assay measurements.

In the last decade the 'comet assay' or 'single cell gel electrophoresis assay' has been established as a sensitive method for the detection of DNA damage and the measurement of its recovery. The results published in the literature have often been obtained with different methods for comet structure measurement. In most cases these data are not comparable with each other. Even when using similar systems for the analysis, it is difficult to obtain matching data. This presentation will describe some technical aspects of our measurement equipment and evaluation software. It focuses on necessary experimental conditions to minimize errors in obtaining such data. The software developed here allows the rapid analysis of the microscopic samples (< 2 s per image). The image analysis was designed with respect to the morphological shapes of comet cells, which were investigated with a confocal laser microscope. The system is built with standard components which are commercially available. As a measure of the amount of DNA damage the ratio of fluorescence intensity was used inside the comet tail and the fluorescence intensity of the comet head. Other parameters such as DNA content, comet area, head radius, tail length and tail moment are also determined. The reproducibility of the system has been evaluated in several experiments over a period of 5 years.

Ataxia Telangiectasia↗

PubMed1996

Comet assay study of DNA damage and repair of tumour cells following boron neutron capture irradiation with fast d(14) + Be neutrons.

We compared the amount of radiation-induced DNA damage and the extent of DNA repair in human melanoma cells (MeWo) using the 'comet assay' after neutron, boron neutron capture and X-irradiation. Using a colony-forming assay it was shown earlier that lethal effects in tumour cells treated with fast neutrons may be increased by the neutron capture reaction 10B(n, alpha)7Li. The effectiveness of boron neutron capture in killing tumour cells depends on the number of 10B atoms delivered to the tumour, the subcellular distribution of 10B and the thermal neutron fluence at the side of the tumour. Using the 'comet assay' the DNA damage of fast neutrons (mean energy 5.8 MeV) was shown to be significantly greater than for the same absorbed dose of X-rays. The presence of 600 ppm 10B (boric acid H5 10BO3) in the cell medium during irradiation with d(14) + Be neutrons in a phantom enhances the DNA damage by 20% compared with neutron irradiation alone. After DNA damage induction by neutrons and neutron capture of boron, the DNA repair capacity of the MeWo cells is significantly reduced in comparison with X-irradiation resulting in proportionally more residual DNA damage after 180 min of repair time.

Boron Neutron Capture Therapy↗

PubMed1996

Comet assay studies indicate that caffeine-mediated increase in radiation risk of embryos is due to inhibition of DNA repair.

It is well known that under specific conditions caffeine is able to enhance radiation risk of mammalian cells by a factor of approximately 1.5-2. Various mechanisms are discussed in the literature as possible explanations for this interaction. Inhibition of DNA repair plays a crucial role in the discussion, although direct evidence for this assumption is difficult to obtain. We used the "comet assay' in order to analyse the significance of repair inhibition by caffeine in the two-cell stage of mammalian gestation. Our data show that at the concentration necessary for increasing radiation risk (2 mM), caffeine effectively inhibits the restitution of radiation-damaged DNA.

Animals↗

PubMed1996

Analysis of DNA damage recovery processes in the adaptive response to ionizing radiation in human lymphocytes.

It has been frequently suggested that the adaptive response to ionizing radiation involves the induction of a chromosomal repair mechanism. Although several lines of evidence favour this assumption, direct proof is lacking. We have chosen to study this question with the help of the comet assay. Lymphocytes from three human donors were given an adapting dose of 0.05 Gy 16 h after mitogenic stimulation and a challenging dose of 2 Gy 5 h thereafter. While a portion of the cells was removed from the cultures for the comet assay, remaining cells were harvested at 52 h culture time and screened for chromosomal aberrations. In some experiments an analysis of cell proliferation was additionally carried out by flow cytometry. In the comet assay a reduced level of initial damage and an increased repair capacity was observed in the adapted + challenged cells; however, this did not result in a reduction of the aberration frequencies. No effect of the adapting dose on cell proliferation was detectable. The analysis of comet distributions revealed that the observed enhanced repair capacity was due to the presence of a subpopulation of slowly repairing cells in the challenged lymphocytes and the lack of such a subpopulation in the adapted + challenged cells. We assume that the slowly repairing cells were quiescent G0 lymphocytes which were removed from the adapted + challenged cell population, probably by apoptotic-like processes.

Adaptation, Physiological↗

PubMed1994

Comet assay studies of radiation-induced DNA damage and repair in various tumour cell lines.

We used the 'comet assay' to compare the amount of radiation-induced DNA damage in three tumour cell lines (MeWo, PECA 4451 and PECA 4197) and the extent of DNA repair in two of these lines (MeWo and PECA 4197). Tumour cells were irradiated with X-rays (0.1-10 Gy), embedded in agarose on slides, lysed with sodium dodecyl sulphate and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depended on the amount of DNA damage. When the cells were incubated at 37 degrees C for various time intervals before electrophoresis started, the comets shrank in the course of time, indicating repair of DNA damage. All three cell lines showed the same extent of DNA damage after radiation exposure, despite the fact that in the colony-forming assay MeWo and PECA 4451 were much more sensitive to radiation exposure than PECA 4197. The repair characteristics, however, were markedly different for MeWo and PECA 4197 cells. PECA 4197 cells showed a much faster restoration of the original shape of the cell nucleus than MeWo cells.

DNA Damage↗

PubMedDate not supplied

[Influence of proles vegetable enzymes on meat].

Studies on the enzymatic digestion of food proteins are being carried out "in vitro" in order to prepare protein hydrolysates to be added to specific diet. The digested product is lyophilized to preserve its solubility characteristics.

Enzyme Activation↗