Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV micrometers-1) no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification.

[Inhibition of 18F-FDG uptake in glioblastoma cells by FDG and glucose]. / Hemmung der 18F-FDG-Aufnahme in Glioblastoma-Zellen durch FDG und Glucose.

It was the aim of the study to compare the inhibition of 18F-2-Fluor-D-deoxy-glucose uptake (18F-FDG) in tumor cells by various concentrations of FDG carrier or D-glucose in an experimental model using tissue culture and positron emission tomography (PET). Glioblastoma cells in culture were incubated with 18F-FDG with and without added carrier or in presence of glucose concentrations in the range from 0-5 mmol/L. Cellular uptake of 18F-FDG was measured after 20 min. of incubation in PBS-buffer containing different sugar concentrations. The uptake was determined with a PET camera. The similarity of the kinetics of the FDG and glucose uptake are backing the hypothesis that both substrates use the same carrier system. The more intense inhibition of the 18F-uptake by FDG can be explained by the different intracellular metabolism of both substrates. The results explain the clinical experience that there is an optimal 18F-FDG uptake in the patient's tumor when the blood glucose level is as low as possible and the specific activity of 18F-FDG is very high.

Increased steady state mRNA levels of DNA-repair genes XRCC1, ERCC2 and ERCC3 in brain of patients with Down syndrome.

Although deficient DNA-repair was proposed for neurodegenerative disorders including Down Syndrome (DS), repair genes for nucleotide excision repair or X-ray repair have not been studied in brain yet. As one of the hypotheses for the pathogenesis of brain damage in DS is oxidative stress and cells of patients with DS are more susceptible to ionizing irradiation, we decided to study ERCC2, ERCC3 and XRCC1, representatives of repair genes known to be involved in the repair of oxidative DNA-damage. mRNA steady state levels of ERCC2, ERCC3, XRCC1, a transcription activator (TAF-DBP) and an elongation factor (EF1A) were determined and normalized versus the housekeeping gene beta-actin in five individual brain regions of nine controls and nine DS patients. Although different in the individual regions, DNA-repair genes were consistently higher in temporal, parietal and occipital lobes of patients with DS accompanied by comparable changes of TFA-DBP and EF1A. Our results are the first to describe DNA-repair gene patterns in human brain regions providing the basis for further studies in this area. We showed that DNA-repair genes ERCC2 and ERCC3 (excision-repair-cross-complementing-) for nucleotide excision repair and XRCC1 (X-ray-repair-cross-complementing-) for X-ray-repair, were increased at the transcriptional level with the possible biological meaning that this increase may be compatible with permanent (oxidative?) DNA damage.

Thyroid stimulating hormone-receptor overexpression in brain of patients with Down syndrome and Alzheimer's disease.

Thyroid hormone abnormalities are strongly associated with Down Syndrome (DS) with elevated thyroid stimulating hormone (TSH) levels as the most consistent finding. Using subtractive hybridization for gene hunting we found significant overexpression of mRNA levels for the TSH-receptor (TSH-R) in brain of a fetus with DS. Based upon this observation we determined TSH-R protein levels in five brain regions of patients with DS (n=8), Alzheimer disease (AD, n=8) and controls (C, n=8). Western blots revealed significantly elevated immunoreactive TSH-R protein(s) 40 kD and 61 kD in temporal and frontal cortex of patients with DS and, unexpectedly, in AD. Levels for the 40 kD protein in temporal cortex were 1.00+/-0.036 (arbitrary units+/-SD) in C, 1.35+/-0.143 in DS, 1.52+/-0.128 in AD; in frontal cortex: 1.00+/-0.046 in C, 1.10+/-0.03 in DS, 1.10+/-0.038 in AD. Levels for the 61 kD protein in temporal cortex were 1.01+/-0.015 in C, 1.47+/-0.013 in DS, 1.623+/-0.026 in AD; in frontal cortex: 1.02+/-0.020 in C, 1.18 +/-0.123 in DS, 1.48+/-0.020 in AD. These results show that elevated brain immunoreactive TSH-R is not specific for DS and maybe reflecting apoptosis, a hallmark of both neurodegenerative disorders, as it is well-documented that the thyroid hormone system is involved in the control of programmed cell death.

Increased phosphoglycerate kinase in the brains of patients with Down's syndrome but not with Alzheimer's disease.

Impaired glucose metabolism in Down's syndrome (DS) has been well-documented in vivo, although information on the underlying biochemical defect is limited and no biochemical studies on glucose handling enzymes have been carried out in the brain. Through gene hunting in fetal DS brain we found an overexpressed sequence homologous to the phosphoglycerate kinase (PGK) gene. This finding was studied further by investigating the activity levels of this key enzyme of carbohydrate metabolism in the brains of patients with DS. PGK activity was determined in five brain regions of nine patients with DS, nine patients with Alzheimer's disease and 14 controls. PGK activity was significantly elevated in the frontal, occipital and temporal lobe and in the cerebellum of patients with DS. PGK activity in corresponding brain regions of patients with Alzheimer's disease was comparable with controls. We conclude that our findings complement previously published data on impaired brain glucose metabolism in DS evaluated by positron emission tomography in clinical studies. Furthermore, we show that in DS, impaired glucose metabolism, represented by increased PGK activity, is a specific finding rather than a secondary phenomenon simply due to neurodegeneration or atrophy. These observations are also supported by data from subtractive hybridization, showing overexpressed PGK in DS brains at the transcriptional level early in life.

Altered gene expression in fetal Down syndrome brain as revealed by the gene hunting technique of subtractive hybridization.

Information on gene expression in brain of patients with Down Syndrome (DS, trisomy 21) is limited and molecular biological research is focussing on mapping and sequencing chromosome 21. The information on gene expression in DS available follows the current concept of a gene dosage effect due to a third copy of chromosome 21 claiming overexpression of genes encoded on this chromosome. Based upon the availability of fetal brain and recent technology of gene hunting, we decided to use subtractive hybridization to evaluate differences in gene expression between DS and control brains. Subtractive hybridization was applied on two fetal brains with DS and two age and sex matched controls, 23rd week of gestation, and mRNA steady state levels were evaluated generating a subtractive library. Subtracted sequences were identified by gene bank and assigned by alignments to individual genes. We found a series of up- and downregulated sequences consisting of chromosomal transcripts, enzymes of intermediary metabolism, hormones, transporters/channels and transcription factors (TFs). We show that trisomy 21 or aneuploidy leads to the deterioration of gene expression and the derangement of transcripts describes the impairment of transport, carriers, channels, signaling, known metabolic and hormone imbalances. The dys-coordinated expression of transcription factors including homeobox genes, POU-domain TFs, helix-loop-helix-motifs, LIM domain containing TFs, leucine zippers, forkhead genes, maybe of pathophysiological significance for abnormal brain development and wiring found in patients with DS. This is the first description of the concomitant expression of a large series of sequences indicating disruption of the concerted action of genes in this disorder.

Gene expression in fetal Down syndrome brain as revealed by subtractive hybridization.

Information on gene expression in brain of patients with Down Syndrome (DS, trisomy 21) is limited and molecular biological research is focussing on mapping and sequencing chromosome 21. The information on gene expression in DS available follows the current concept of a gene dosage effect due to a third copy of chromosome 21 claiming overexpression of genes encoded on this chromosome. Based upon the availability of fetal brain and recent technology of gene hunting, we decided to use subtractive hybridization to evaluate differences in gene expression between DS and control brains. Subtractive hybridization was applied on two fetal brains with DS and two age and sex matched controls, 23rd week of gestation, and mRNA steady state levels were evaluated generating a subtractive library. Subtracted sequences were identified by gene bank and assigned by alignments to individual genes. We found a series of up- and downregulated sequences consisting of chromosomal transcripts, enzymes of intermediary metabolism, hormones, transporters/channels and transcription factors (TFs). We show that trisomy 21 or aneuploidy leads to the deterioration of gene expression and the derangement of transcripts described describes the involvement of chromosomes other than chromosome 21, explains impairment of transport, carriers, channels, signaling, known metabolic and hormones imbalances. The dys-coordinated expression of transcription factors including homeobox genes, POU-domain TFs, helix-loop-helix-motifs, LIM domain containing TFs, leucine zippers, forkhead genes, maybe of pathophysiological significance for abnormal brain development and wiring found in patients with DS. This is the first description of the concomitant expression of a large series of sequences indicating disruption of the concerted action of genes in that disorder.

Overexpression of DNAse I in brain of patients with Down syndrome.

Human DNAse I (EC 3.1.21.1) is an enzyme most probably involved in apoptotic processes. Splicing of the DNAse I primary transcript in normal and apoptotic cells into up to 20 splicing forms and the recent description of a different family of caspase-activated DNAses, hampered studies on the role of DNAse I in apoptosis research. Performing gene hunting in fetal brain of patients with DS we found a sequence with 100% homology to DNAse I and this formed the Rationale for studies in adult DS brain. It was therefore the aim of the study to evaluate DNAse I-mRNA steady state levels in DS brain using adult brain without brain pathologies and Alzheimer's Disease (AD) brain as control, in order to rule out that DNAse I--overexpression may not be specific for DS but rather reflecting apoptosis per se, a hallmark of both disorders. Determination of DNAse I-mRNA steady state levels was carried out by a blotting method in frontal, parietal, temporal occipital lobe and cerebellum. We found significantly increased DNAse I transcripts in brain of DS and AD both, when normalized versus the house-keeping gene beta actin or total RNA. We demonstrate the significant increase of DNAse I--transcript in the pathogenesis of DS and AD suggesting a role for this enzyme in the apoptotic process known to occur in both disorders. We are now going to carry out protein and enzyme activity levels in our laboratory to confirm our findings at the transcriptional level.

Evidence that taurine modulates osmoregulation by modification of osmolarity sensor protein ENVZ--expression.

Although the involvement of taurine in osmoregulation is well-documented and widely accepted, no detailed mechanism for this function has been reported so far. We used subtractive hybridization to study mRNA steady state levels of genes up- or downregulated by taurine. Rats were fed taurine 100 mg/kg body weight per day for a period of three days and hearts (total ventricular tissue) of experimental animals and controls were pooled and used for mRNA extraction. mRNAs from two groups were used for subtractive hybridization. Clones of the subtractive library were sequenced and the obtained sequences were identified by gen bank assignment. Two clones were found to contain sequences which could be assigned to the osmolarity sensor protein envZ, showing homologies of 61 and 65%. EnvZ is an inner membrane protein in bacteria, important for osmosensing and required for porine gene regulation. It undergoes autophosphorylation and subsequently phosphorylates OmpR, which in turn binds to the porine (outer membrane protein) promoters to regulate the expression of OmpF and OmpC, major outer membrane porines. This is the first report of an osmosensing mechanism in the mammalian system, which was described in bacteria only. Furthermore, we are assigning a tentative role for taurine in the osmoregulatory process by modifying the expression of the osmoregulatory sensor protein ENVZ.

Decreased transcription factor junD in brains of patients with Down syndrome.

JunD is a member of the Jun family of transcription factors (TF), recently shown to negatively regulate cell growth and antagonizes transformation by the protooncogene ras: c-jun decreases while junD is accumulating when fibroblasts become quiescent. Furthermore, overexpression of junD resulted in slower growth and an increase in cells in G0/G1. Performing gene hunting on fetal Down syndrome (DS) brain we found a sequence downregulated and homologous to junD. This observation made us examine junD protein levels in adult brain specimens. Western blot experiments were carried out in five brain regions of aged patients with DS (n = 9), controls (n = 9) and patients with Alzheimer's disease (AD, n = 9). We found that junD in AD brains were comparable to controls, whereas junD levels were significantly and remarkably reduced in frontal, temporal lobe and cerebellum of patients with DS. These findings may indicate a specific finding in DS and were not linked to the AD-like-neuropathological changes of plaques and tangles, observed in DS from the fourth decade, which is also suggested by the findings of downregulated junD at the mRNA level revealed by the gene hunting technique (subtractive hybridization) in fetal DS brain. We propose that junD plays a role for the impaired development and wiring of DS brain, maybe already early in life.

Proton magnetic resonance spectroscopy of the primary motor cortex in patients with motor neuron disease: subgroup analysis and follow-up measurements.

OBJECTIVES: To determine the motor cortex degeneration in patients with amyotrophic lateral sclerosis (ALS) using proton magnetic resonance spectroscopy, and to prove that proton magnetic resonance spectroscopy is suited to monitor the course of disease with follow-up examinations. MATERIALS AND METHODS: We studied 33 patients with ALS whose conditions were diagnosed according to the El Escorial World Federation of Neurology criteria. Nine patients with ALS were followed up for up to 2 years. The control group included 20 healthy volunteers and 4 patients with multifocal motor neuropathy. Proton magnetic resonance spectroscopy determined levels of the brain metabolites N-acetylaspartate (NAA), choline, inositol-containing compounds, glutamate/glutamine, and phosphocreatine. RESULTS: Patients with ALS showed a significant reduction in the NAA-choline (P <.001) and NAA-phosphocreatine (P <.005) metabolite ratios and significantly elevated choline-phosphocreatine (P <.005) ratios compared with controls. Inositol-phosphocreatine ratios were also elevated in case patients, but the increase was less pronounced (P <.05). No differences in glutamate/glutamine-phosphocreatine ratios were detected between case patients and controls. An analysis of subgroups demonstrated less significant differences in NAA-choline metabolite ratios (P<.05), even in patients with pure lower motor neuron syndrome (suspected ALS). No changes in metabolite T1 and T2 relaxation times were observed. Patients with multifocal motor neuropathy showed normal metabolic ratios. Progressive alterations in affected metabolite ratios could be documented in the follow-up examinations. CONCLUSIONS: Spectroscopic changes in the motor cortices of patients with ALS correspond with a reduction in levels of NAA and an elevation in levels of choline and inositol compounds. Since NAA is exclusively expressed in neurons, the observed decrease of NAA reflects neuronal loss or dysfunction. Inositol and choline are associated with plasma membrane metabolism, so the release of these compounds may be related to membrane disorders.

Transcription of the XRCC1 gene in kidneys of radiosensitive and radioresistant mice following whole-body irradiation.

The XRCC1 gene was described to play a role in the sensitivity of mammalian cell lines towards ionizing irradiation. Cells with a mutation of this gene present with decreased single-strand break repair and reduced recombination repair, show increased double-strand breaks, and the sister chromatid exchange is increased up to tenfold. The goal of our study was to investigate the transcription of this gene in the kidney following ionizing irradiation in the mouse, as this could be relevant to the pathogenetic mechanisms found in radiation nephropathy. Furthermore, we intended to examine whether radiation-sensitive mice would show a transcriptional pattern different from radiation-resistant mice. Radiation-sensitive BALB/c/J/Him mice and radiation-resistant C3H/He/Him mice were whole body irradiated with X-ray at 2, 4, and 6 Gy and sacrificed 5, 15, and 30 min after irradiation. mRNA was isolated from kidney cortex and hybridized with probes for XRCC1 and beta-actin as a housekeeping gene control. Following irradiation at 2 Gy, radiation-resistant mice increased transcriptional levels of mRNA-XRCC1/mRNA-beta-actin as early as after 5 min, and 15 and 30 min after irradiation, XRCC1 transcription was still higher than in radiation-sensitive mice. At higher radiation doses, no differences were found. This finding is the first in vivo study on XRCC1 of this kind and may in part explain the differences in the radiation sensitivity between the two strains studied.

[31P-mr spectroscopy of peripheral skeletal musculature under load: demonstration of normal energy metabolites compared with metabolic muscle diseases]. / 31P-MR-Spektroskopie der peripheren Skelettmuskulatur unter Belastung: Darstellung des normalen Energiestoffwechsels im Vergleich zu metabolischen Muskelerkrankungen.

PURPOSE: 31P-MR spectroscopy of skeletal muscle under exercise was used to obtain the range of normal variation and comparison was made for different neuromuscular diseases. METHODS: 41 examinations of 24 volunteers and 41 investigations in 35 patients were performed on 1.5 T MR systems (Gyroscan 515 und S15/ACSII, Philips). Localised 31P-MR spectra of the calf muscle were obtained in time series with a resolution of 12 s. RESULTS: Two types of muscle energy metabolism were identified from the pattern of spectroscopic time course in volunteers: While the first group was characterised by a remarkable decline to lower pH values during exercise, the second group showed only small pH shifts (minimum pH: 6.48 +/- 0.13 vs 6.87 +/- 0.07, p < 10(-6)) although comparable workload conditions were maintained. The pH-values correlated well with blood lactate analysis. Patients with metabolic disorders and chronic fatigue syndrome (CFS) showed decreased resting values of PCr/(PCr + Pi) and increased pH levels during exercise. PCr recovery was significantly delayed (0.31 vs 0.65 min-1, p < 0.00005) in metabolic muscle disorders but was normal in CFS patients. CONCLUSION: Findings in volunteers indicate utilisation of different metabolic pathways which seems to be related to the fibre type composition of muscle. Reduced resting levels for PCr/(PCr + Pi), altered pH time courses, and decreased PCr recovery seem to be helpful indicators for diagnosis of metabolic muscle disorders.

Computer modeling of cytokeratin release in clinical oncology.

The levels of cytokeratins (CK) in serum of cancer patients have been widely used for monitoring progression of cancer growth and the effectiveness of cancer treatment. Previous studies have shown that the release of CK by tumors in patients is a complex process which depends on the rate of cell damage caused by an increasing tumor mass, or by the tumor treatment, but is not in any simple manner correlated to the number of proliferating cells or to the total tumor mass (1). The complexity of the CK-releasing process has been analyzed by a computer model which mimics the progress of tumor growth, allows the introduction of different types of treatment (i.e. irradiation, chemotherapy and surgery), and computes the amount of CK released by the tumor, and the level of CK in blood and blood clearance. The computer model can be used to obtain a better understanding of the interactions of various factors, for scheduling of treatment and CK sampling, and for analyzing the effects of treatment.

The transcription of liver thioredoxin following the ionizing irradiation of radioresistant and radiosensitive mice.

The radiation protective effect of thioredoxin (TRX) in a bacterial system has been reported and based upon this observation we were interested to examine TRX transcription in the mammalian system following ionizing irradiation. In order to answer the question whether radiation sensitive mice (BALB/c) showed TRX transcription different from radiation resistant mice (C3H), we exposed these strains to X-ray doses of 2 Gy, 4 Gy and 6 Gy. Groups consisting of 6 mice were sacrificed 5, 15 and 30 minutes after irradiation and livers were immediately taken into liquid nitrogen. Total RNA was isolated from the organs by the use of a commercially available kit and used for Northern blots and slot blots with a chemiluminescence technique. Northern blots revealed a single band at 538 bp for TRX and at 1.8 kb for beta-actin. Quantification of mRNA TRX by densitometry of slot blots revealed that C3H transcribed TRX significantly higher at an earlier time point (5 min) than BALB/c. This delayed transcription of TRX in the radiosensitive mouse strain showed a comparable pattern at three different radiation doses and may well be responsible for radioresistance although no quantitative differences of TRX transcription between BALB/c and C3H mice were detectable.

The influence of microgravity on repair of radiation-induced DNA damage in bacteria and human fibroblasts.

The influence of the space flight environment, above all microgravity, on the repair of radiation-induced DNA damage was examined during the Spacelab mission IML-2 as (1) rejoining of DNA strand breaks induced by X irradiation in cells of Escherichia coli B/r (120 Gy) and (2) in human fibroblasts (5 and 10 Gy); (3) induction of the SOS response after gamma irradiation (300 Gy) of cells of Escherichia coli PQ37; and (4) survival of spores of Bacillus subtilis HA 101 after UV irradiation (up to 340 J m(-2)). Cells were irradiated prior to the space mission and were kept frozen (E. coli and fibroblasts) until incubation for defined periods (up to 4.5 h) in orbit; thereafter they were frozen again for laboratory analysis. Germination and growth of spores of B. subtilis on membrane filters was initiated by humidification in orbit. Controls were performed in-flight (1g reference centrifuge) and on the ground (1g and 1.4g). We found no significant differences between the microgravity samples and the corresponding controls in the kinetics of DNA strand break rejoining and of the induction of the SOS response as well as in the survival curves (as proven by Student's t test, P < or = 0.1). These observations provide evidence that in the microgravity environment cells are able to repair radiation-induced DNA damage almost normally. The results suggest that a disturbance of cellular repair processes in the microgravity environment might not be the explanation for the reported synergism of radiation and microgravity.

The transcription of the XRCC1 gene in the heart of radiation-resistant and radiation-sensitive mice after ionizing irradiation.

The XRCC1 (X-Ray Repair Cross Complementing) gene was described to play a role for the sensitivity of mammalian cell lines toward ionizing irradiation. Cells with a mutation of this gene present with decreased single strand break repair and reduced recombination repair, they show increased double strand breaks, and sister chromatid exchange is increased up to 10-fold. The goal of our study was to investigate the transcription of this gene in the heart after ionizing irradiation in the mouse. Furthermore, we intended to examine whether radiation-sensitive mice would show a transcriptional pattern different from radiation-resistant mice. Radiation-sensitive BALB/c/J Him mice and radiation-resistant C3H He/Him mice were whole body irradiated with x-ray at 2, 4, and 6 Gy and killed 5, 15, and 30 min after irradiation. mRNA was isolated from the heart and hybridized with probes for XRCC1 and beta-actin as a housekeeping gene control. Irradiation at 2 Gy showed increased transcription of XRCC1 at 5 min in the C3H He/Him group, approached XRCC1 transcription of BALB/c J/Him mice at 15 min, and was lower in the latter at 30 min after irradiation. Irradiation at 4 Gy showed double the transcription at 5 min and an about 3-fold rapid increase of mRNA XRCC1 in the radiation-resistant group at 15 min after irradiation, returning to the transcriptional level of sensitive animals at 30 min. Irradiation at 6 Gy seemed to overwhelm the system in both groups, but resistant mice still showed higher levels of XRCC1 transcription. We conclude that radiation-resistant mice show a higher transcription level for the XRCC1 gene in the heart early after x-ray whole body irradiation. This findings is the first in vivo study on XRCC1 of this kind and may in part explain the differences in the radiation sensitivity between the two strains studied.

The transcription of the XRCC1 gene in spleen following ionizing irradiation in radiosensitive and radioresistant mice.

The XRCC1 gene was described to play a role for the sensitivity of mammalian cell lines towards ionizing irradiation. Cells with a mutation of this gene present with decreased single strand break repair, reduced recombination repair, show increased double strand breaks and sister chromatid exchange is increased up to tenfold. The goal of our study was to investigate the transcription of this gene in the spleen following ionizing irradiation in the mouse. Furthermore, we intended to examine whether radiation sensitive (RS) mice would show a transcriptional pattern different from radiation resistant (RR) mice. Radiation sensitive BALB/c/J Him mice and radiation resistant C3H He/Him mice were untreated or whole body irradiated with X-ray at 4 and 6 Gy and sacrificed 5, 15 and 30 min after irradiation. mRNA was isolated from the spleen and hybridized with probes for XRCC1 and beta-actin as a house keeping gene control. Transcription of XRCC1 was not different in unirradiated or 4 Gy-irradiated mouse RR or RS mouse strains. When irradiated at 6 Gy, RR mice showed an approximately threefold increase of mRNA XRCC1/mRNA beta actin as early as 15 min after irradiation. We conclude that radiation resistant mice show a higher transcription level for the XRCC1 gene in the spleen early after high dose X-ray whole body irradiation. This finding is the first in vivo study on XRCC1 of this kind and may in part explain the differences in the radiation sensitivity between the two strains studied.

DNA repair in microgravity: studies on bacteria and mammalian cells in the experiments REPAIR and KINETICS.

The impact of microgravity on cellular repair processes was tested in the space experiments REPAIR and KINETICS, which were performed during the IML-2 mission in the Biorack of ESA: (a) survival of spores of Bacillus subtilis HA101 after UV-irradiation (up to 340 J m-2) in the experiment REPAIR; (b) in the experiment KINETICS the kinetics of DNA repair in three different test systems: rejoining of X-ray-induced DNA strand breaks (B1) in cells of Escherichia coli B/r (120 Gy) and (B2) in human fibroblasts (5 and 10 Gy) as well as (B3) induction of the SOS response after gamma-irradiation (300 Gy) of cells of Escherichia coli PQ37. Cells were irradiated prior to the space mission and were kept in a non-metabolic state (metabolically inactive spores of B. subtilis on membrane filters, frozen cells of E. coli and human fibroblasts) until incubation in orbit. Germination and growth of B. subtilis were initiated by humidification, E. coli and fibroblasts were thawed up and incubated at 37 degrees C for defined repair periods (up to 4.5 h), thereafter they were frozen again for laboratory analysis. Relevant controls were performed in-flight (1 x g reference centrifuge) and on ground (1 x g and 1.4 x g) The results show no significant differences between the microgravity samples and the corresponding controls neither in the survival curves nor in the kinetics of DNA strand break rejoining and induction of the SOS response (proven by Student's t-test, 2 P = 0.05). These observations provide evidence that in the microgravity environment cells are able to repair radiation-induced DNA damage close to normality. The results suggest that a disturbance of cellular repair processes in the microgravity environment might not be the explanation for the reported synergism of radiation and microgravity.

A new combination of protecting groups and links for encoded synthetic libraries suited for consecutive tests on the solid phase and in solution.

A strategy for high-throughput evaluation of combinatorial compound libraries is reported, which circumvents the necessity to test complex mixtures. The method is based on a new combination of protecting groups, solid-phase linker and tags. The bulk of the library first undergoes a binding assay with the components grafted on beads. A selection of beads carrying strong ligands is stripped from the labelled target and distributed into microvessels. The ligands are cleaved and rinsed into microeluates. Subsequently, a more detailed characterization with a functional assay in solution determines the best performers, which are identified through the peptidic tag left behind on the corresponding mother bead.