The European radiobiological archives: online access to data from radiobiological experiments.

For financial and ethical reasons, the large-scale radiobiological animal studies conducted over the past 50 years are, to a large extent, unrepeatable experiments. It is therefore important to retain the primary data from these experiments to allow reanalysis, reinterpretation and re-evaluation of results from, for example, carcinogenicity studies, in the light of new knowledge in radiation biology. Consequently, there is an imperative need to keep these data available for the research community. The European Radiobiological Archives (ERA) were developed to fulfill this task. ERA has become a unique archive, including information from almost all European long-term studies carried out between the 1960s and the 1990s. The legacy database was originally developed in a manner that precluded online use. Therefore, strong efforts were made to transform it into a version that is available online through the web. This went together with quality assurance measures, including first the estimation of the rate of non-systematic errors in data entry, which at 2% proved to be very low. Second, every data set was compared against two external sources of information. Standardization of terminology and histopathology is a prerequisite for meaningful comparison of data across studies and analysis of potential carcinogenic effects. Standardization is particularly critical for the construction of a database that includes data from different studies evaluated by pathologists in different laboratories. A harmonized pathology nomenclature with modern standard pathology terms was introduced. As far as possible, references for the various studies were directly linked to the studies themselves. Further, a direct link to the JANUS database was established. ERA is now in a position where it has the potential to become a worldwide radiobiological research tool. ERA can be accessed at no cost at https://era.bfs.de. An ID and password can be obtained from the curators at era@bfs.de .

Review and meta-analysis of epidemiological associations between low/moderate doses of ionizing radiation and circulatory disease risks, and their possible mechanisms.

Although the link between high doses of ionizing radiation and damage to the heart and coronary arteries has been well established for some time, the association between lower-dose exposures and late occurring cardiovascular disease has only recently begun to emerge, and is still controversial. In this paper, we extend an earlier systematic review by Little et al. on the epidemiological evidence for associations between low and moderate doses of ionizing radiation exposure and late occurring blood circulatory system disease. Excess relative risks per unit dose in epidemiological studies vary over at least two orders of magnitude, possibly a result of confounding and effect modification by well-known (but unobserved) risk factors, and there is statistically significant (p < 0.00001) heterogeneity between the risks. This heterogeneity is reduced, but remains significant, if adjustments are made for the effects of fractionated delivery or if there is stratification by endpoint (cardiovascular disease vs. stroke, morbidity vs. mortality). One possible biological mechanism is damage to endothelial cells and subsequent induction of an inflammatory response, although it seems unlikely that this would extend to low-dose and low-dose-rate exposure. A recent paper of Little et al. proposed an arguably more plausible mechanism for fractionated low-dose effects, based on monocyte cell killing in the intima. Although the predictions of the model are consistent with the epidemiological data, the experimental predictions made have yet to be tested. Further epidemiological and biological evidence will allow a firmer conclusion to be drawn.

Progress in updating the European Radiobiology Archives.

PURPOSE: The European Radiobiology Archives (ERA), together with corresponding Japanese and American databases, hold data from nearly all experimental animal radiation biology studies carried out between 1960 and 1998, involving more than 300,000 animals. The Federal Office for Radiation Protection, together with the University of Cambridge have undertaken to transfer the existing ERA archive to a web-based database to maximize its usefulness to the scientific community and bring data coding and structure of this legacy database into congruence with currently accepted semantic standards for anatomy and pathology. METHODS: The accuracy of the primary data input was assessed and improved. The original rodent pathology nomenclature was recoded to replace the local 'DIS-ROD' (Disease Rodent) formalism with Mouse Pathology (MPATH) and Mouse Anatomy (MA) ontology terms. A pathology panel sampled histopathological slide material and compared the original diagnoses with currently accepted diagnostic criteria. RESULTS: The overall non-systematic error rate varied among the studies between 0.26% and 4.41%, the mean error being 1.71%. The errors found have been corrected and the studies thus controlled have been annotated. The majority of the original pathology terms have been successfully translated into a combination of MPATH and MA ontology terms. CONCLUSIONS: ERA has the potential of becoming a world-wide radiobiological research tool for numerous applications, such as the re-analysis of existing data with new approaches in the light of new hypotheses and techniques, and using the database as an information resource for planning future animal studies. When the database is opened for new data it may be possible to offer long-term storage of data from recent and future animal studies.

Prognostic value of protein tyrosine kinase 6 (PTK6) for long-term survival of breast cancer patients.

The cytoplasmic tyrosine kinase PTK6 (BRK) shows elevated expression in approximately two-thirds of primary breast tumours, and is implicated in EGF receptor-dependent signalling and epithelial tumorigenesis. Using immunohistochemistry, we performed a retrospective study on 426 archival breast cancer samples from patients with long-term follow-up and compared the protein expression levels of PTK6, the HER receptors, Sam68 (a substrate of PTK6), and signalling proteins including MAP kinase (MAPK), phosphorylated MAPK (P-MAPK), and PTEN. We show that PTK6 expression is of significant prognostic value in the outcome of breast carcinomas. In multivariate analysis, the disease-free survival of patients of >or=240 months was directly associated with the protein expression level of PTK6 (P

A systematic review of epidemiological associations between low and moderate doses of ionizing radiation and late cardiovascular effects, and their possible mechanisms.

Little, M. P., Tawn, E. J., Tzoulaki, I., Wakeford, R., Hildebrandt, G., Paris, F., Tapio, S. and Elliott, P. A Systematic Review of Epidemiological Associations Between Low and Moderate Doses of Ionizing Radiation and Late Cardiovascular Effects, and Their Possible Mechanisms. Radiat. Res. 169, 99-109 (2008). The link between high doses of ionizing radiation and damage to the heart and coronary arteries is established. In this paper, we systematically review the epidemiological evidence for associations between low and moderate doses (<5 Gy) of ionizing radiation and late-occurring cardiovascular disease. Risks per unit dose in epidemiological studies vary over at least two orders of magnitude, possibly a result of confounding factors. An examination of possible biological mechanisms indicates that the most likely causative effect of radiation exposure is damage to endothelial cells and subsequent induction of an inflammatory response, although it seems unlikely that this would extend to low-dose and low-dose-rate exposure. However, a role for somatic mutation has been proposed that would indicate a stochastic effect. In the absence of a convincing mechanistic explanation of epidemiological evidence that is less than persuasive at present, a cause-and-effect interpretation of the reported statistical associations cannot be reliably inferred, although neither can it be reliably excluded. Further epidemiological and biological evidence will allow a firmer conclusion to be drawn.

Stapes surgery: a 32-year follow-up.

Despite a widespread use of stapes surgery, little is known about the long-term durability of hearing results. The present study provides data over a long time frame (32 years) on hearing changes following surgical treatment. During a 10-year period (1965-1975) stapes surgery was performed in 322 consecutive patients in Tampere University Hospital, Finland. Postoperative evaluation was performed in the same hospital during the years 2001 and 2002 in 58 patients (41 females, 17 males). Of these 58 patients, 68 ears were operated decades ago (stapedoplasty in 67 ears and stapes mobilization in one ear). The mean follow-up time at the latest visit was 32.9 years. At the long-term follow-up, we found that the operative result was very well maintained: 37% of the patients had air-bone gap closure less than 10 dB. Most of the patients (83%) reached postoperative (6-12 months) air-bone gap closure less than 20 dB. The result was maintained by 75% of the patients. Stapes surgery gives a rise to a better social life for the hearing-impaired patient and delays the need for hearing aid in most patients.

Starvation in vivo for aminoacyl-tRNA increases the spatial separation between the two ribosomal subunits.

Structures in situ of individual ribosomes in E. coli have been determined by computer-aided electron microscope tomography using a tilt series of positively stained embedded cellular sections. Amino acid starvation of a bacterial culture, causing a deficiency for aminoacyl-tRNA, induces a spatial separation between the ribosomal subunits compared with ribosomes in exponentially growing cells. Eight ribosomes from each growth condition were aligned to each other, and the two average structures were determined. Comparison of these suggests that the distance between the two subunits increases by approximately 3 nm upon starvation for aminoacyl-tRNA during protein synthesis. Ribosomes in most other states of the translational elongation cycle in exponentially growing cells show a more compact structure than previously realized.

Molecular characterization of the MalT-dependent periplasmic alpha-amylase of Escherichia coli encoded by malS.

malS, the gene encoding the periplasmic alpha-amylase, is under the regulatory control of the MalT protein, the gene activator of the Escherichia coli maltose system. We sequenced the DNA region encoding malS and its control elements. malS consists of an open reading frame of 2,028 base pairs encoding a protein of 676 amino acids with a deduced molecular weight of 75,664 including a typical amino-terminal signal sequence of 17 amino acids. The deduced amino acid sequence of malS was compared with that of other proteins. We found homologies to alpha-amylases and a cyclodextrin glucanotransferase but not to beta-amylases. In addition, MalS showed significant homology to another maltodextrin-utilizing and MalT-dependent enzyme of E. coli, maltodextrin glucosidase (MalZ) but not to amylomaltase (MalQ), the major maltodextrin-degrading enzyme. Conserved regions that have been proposed to constitute enzymatically active sites in alpha-amylases are present in MalS. Two of these sequences can also be found in the amino terminus of the lambda-receptor, a maltodextrin-specific channel in the outer membrane. The nucleotide sequence of the control region of malS revealed MalT binding sites correctly spaced for the start of the malS transcript. At the position 219 base pairs upstream of malS, we found a divergently transcribed gene, bax, which has been recognized previously. Downstream of malS, after a 513-base pair intergenic region, lies the convergently transcribed gene avtA, which codes for the alanine/valine transaminase.

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.

The malZ gene of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase.

We have characterized a maltodextrin glucosidase, previously described as a maltose-inducible, cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside in Escherichia coli. The gene encoding the enzyme activity, referred to as malZ, is located at 9.3 min on the chromosomal map. We cloned the gene in a high copy number vector and purified the enzyme. It is a monomer, with an apparent molecular weight of 65,000. The enzyme degrades maltodextrins, ranging from maltotriose to maltoheptaose, to shorter oligosaccharides, the final hydrolysis products being maltose and glucose. We measured the kinetic parameters, Km and Vmax, for the hydrolysis to glucose of the five different substrates. The binding of the substrate is enhanced by increasing the number of glucosyl residues in the maltodextrin. In contrast, the maximum rate of hydrolysis (Vmax) is fastest for maltotriose. To study the mode of action of the enzyme, we quantitatively measured the amount of free glucose liberated from the different maltodextrin substrates after a long incubation. More glucose is liberated from the long dextrins, as compared to the shorter ones, showing that the primary hydrolysis product was glucose, not maltose. Furthermore, [14C]maltotriose, specifically labeled at the reducing end, was hydrolyzed to [14C]glucose and unlabeled maltose. These data demonstrate that the malZ gene product is a maltodextrin glucosidase, liberating glucose from the reducing end of malto-oligosaccharides. The nucleotide sequence of malZ and the deduced amino acid sequence showed that malZ encodes a protein with a molecular weight of 68,960. Homology to glucosidases, alpha-amylases, and pullulanases were observed. Conserved regions thought to represent active sites in dextrin hydrolases were found in the MalZ protein.

Antisuppression by mutations in elongation factor Tu.

Two slow-growing kirromycin-resistant Escherichia coli mutants with altered EF-Tu (Ap and Aa) were studied in vivo in strains with an inactive tufB gene. Mutant form Aa was isolated as an antisuppressor of the tyrT(Su3) nonsense suppressor, as described here. Ap, the tufA gene product of strain D2216 (from A. Parmeggiani), has previously been shown to give an increased GTPase activity. The slow cellular growth rates of both EF-Tu mutants are correlated with decreased translational elongation rates. Ap and Aa significantly decrease suppression levels of both nonsense and missense suppressor tRNAs [tyrT(Su3), trpT(Su9), glyT(SuAGA/G)], but have only little or no effect on misreading by wild-type tRNAs. A particular missense suppressor, lysT(SuAAA/G), which acts by virtue of partial mischarging as the result of an alteration in the amino acid stem, is not significantly affected by the EF-Tu mutations. The combination of tufA(Aa) and a rpsD12 ribosomal mutation is lethal at room temperature and the double-mutant strain has an elevated temperature optimum (42 degrees C) for growth rate, translation rate and nonsense suppression. Our data indicate an alterated interaction between Aa and the ribosome, consistent with our in vitro results.

Impaired in vitro kinetics of EF-Tu mutant Aa.

The kirromycin-resistant EF-Tu mutant Aa, previously shown to be an antisuppressor for nonsense and missense suppressor tRNAs, has been characterised in a poly(U)-primed translation system in vitro. Two major defects were found in the function of the mutant. First, the dissociation constant for Aa binding to Phe-tRNA(Phe) was increased tenfold compared to wild-type EF-Tu. Second, kcat/Km for the interaction between the EF-Tu.GTP.aa-tRNA complex and the ribosome was decreased by the mutation to one third of its wild-type value. No differences were observed between mutant and wild-type factor in the regeneration of EF-Tu.GTP from EF-Tu.GDP via EF-Ts or in the mistranslation frequency by Leu-tRNA(4Leu). The relation between the in vitro results and the mutant phenotype in vivo is discussed.

Antagonistic effects of mutant elongation factor Tu and ribosomal protein S12 on control of translational accuracy, suppression and cellular growth.

Kirromycin-resistant mutant forms of elongation factor Tu, which are coded by tufA (Ar) or tufB (Bo) and are associated with an increased rate of translational error formation, have been analysed. In vivo, Ar was found to increase misreading as well as suppression of non-sense codons irrespective of Bo in a strain with wild type ribosomes. It is therefore not necessary to evoke both tufA (Ar) and tufB (Bo) mutations together in order to increase translational error as suggested earlier [1]. When combined with a hyperaccurate ribosomal rpsL (S12) mutation, Ar counteracts the restrictive effects on translational error formation caused by the altered protein S12, thus restoring the levels of missense error in vitro and non-sense error and suppression in vivo to near wild type values. As judged from in vitro experiments this results principally from a lowered selectivity of the Ar ternary complex at the initial discrimination step on the ribosome during translation. In vivo, this compensatory effect on the rpsL mutation on non-sense error formation and suppression is seen irrespective of the nature of tRNA or codon context. Furthermore, the tufA mutation enhances the cellular growth rate of the rpsL mutant, whereas it decreases growth of strains with normal ribosomes. Inactivation of one of the two genes coding for EF-Tu (tufB), while leaving the other gene (tufA) intact, can by itself, increase non-sense error formation and suppression.

Mutant EF-Tu increases missense error in vitro.

We have studied the consequences of mutational alteration in the structure of EF-Tu on the missense errors and proofreading activity of bacterial ribosomes in vitro. Our data show that the EF-Tu Bo mutant form of EF-Tu (van der Meide et al. 1983a) is inactive in polypeptide synthesis on the ribosome, even though it binds aminoacyl-tRNA. A second mutant form, EF-Tu Ar (van der Meide et al. 1983a), is active in polypeptide synthesis but supports a much higher messense incorporation with either leucine isoacceptor 2 or leucine isoacceptor 4 in the in vitro system. Further analysis of the kinetic basis of this enhanced missense frequency revealed that the mutation responsible for the alteration in EF-Tu Ar increases the errors at both the proofreading step and the initial selection. In this respect the effect of this particular mutation is similar to the mode of action of the antibiotic kanamycin (Jelenc and Kurland 1984).