Genotoxic risk of ethyl-paraben could be related to telomere shortening.

The ability of parabens to promote the appearance of multiple cancer hallmarks in breast epithelium cells provides grounds for regulatory review of the implication of the presence of parabens in human breast tissue. It is well documented that telomere dysfunction plays a significant role in the initiation of genomic instability during carcinogenesis in human breast cancer. In the present study, we evaluated the genotoxic effect of ethyl 4-hydroxybenzoate (ethyl-paraben), with and without metabolic activation (S9), in studies following OECD guidelines. We observed a significant increase in genotoxic damage using the Mouse Lymphoma Assay and in vitro micronucleus (MN) tests in the L5178Y cell line in the presence of S9 only after a short exposure. A high frequency of MN was observed in the TK6 cells after a short exposure (3 h) in the presence of S9 and a long exposure (26 h) without S9. We found significant increases in the MN frequency and induced chromosomal aberrations in the lymphocytes of only one donor after ethyl-paraben exposure in the presence of S9 after a short exposure. Cytogenetic characterization of the paraben-treated cells demonstrated telomere shortening associated with telomere loss and telomere deletions in L5178Y and TK6 cells and lymphocytes of the paraben sensitive-donor. In a control cohort of 68 human lymphocytes, telomere length and telomere aberrations were age-dependent and showed high inter-individual variation. This study is the first to link telomere shortening and the genotoxic effect of ethyl paraben in the presence of S9 and raises the possibility that telomere shortening may be a proxy for underlying inter-individual sensitivity to ethyl-paraben. Copyright © 2016 John Wiley & Sons, Ltd.

Chromatin remodeling by the T cell receptor (TCR)-beta gene enhancer during early T cell development: Implications for the control of TCR-beta locus recombination.

Gene targeting studies have shown that T cell receptor (TCR)-beta gene expression and recombination are inhibited after deletion of an enhancer (Ebeta) located at the 3' end of the approximately 500-kb TCR-beta locus. Using knockout mouse models, we have measured, at different regions throughout the TCR-beta locus, the effects of Ebeta deletion on molecular parameters believed to reflect epigenetic changes associated with the control of gene activation, including restriction endonuclease access to chromosomal DNA, germline transcription, DNA methylation, and histone H3 acetylation. Our results demonstrate that, in early developing thymocytes, Ebeta contributes to major chromatin remodeling directed to an approximately 25-kb upstream domain comprised of the Dbeta-Jbeta locus regions. Accordingly, treatment of Ebeta-deleted thymocytes with the histone deacetylase inhibitor trichostatin A relieved the block in TCR-beta gene expression and promoted recombination within the Dbeta-Jbeta loci. Unexpectedly, however, epigenetic processes at distal Vbeta genes on the 5' side of the locus and at the 3' proximal Vbeta14 gene appear to be less dependent on Ebeta, suggesting that Ebeta activity is confined to a discrete region of the TCR-beta locus. These findings have implications with respect to the developmental control of TCR-beta gene recombination, and the process of allelic exclusion at this locus.

The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor.

Transcription of the 45S rRNA genes is carried out by RNA polymerase I and at least two trans-acting factors, upstream binding factor (UBF) and SL-1. We have examined the hypothesis that SL-1 and UBF interact. Coimmunoprecipitation studies using an antibody to UBF demonstrated that TATA-binding protein, a subunit of SL-1, associates with UBF in the absence of DNA. Inclusion of the detergents sodium dodecyl sulfate and deoxycholate disrupted this interaction. In addition, partially purified UBF from rat cell nuclear extracts and partially purified SL-1 from human cells coimmunoprecipitated with the anti-UBF antibody after mixing, indicating that the UBF-SL-1 complex can re-form. Treatment of UBF-depleted extracts with the anti-UBF antibody depleted the extracts of SL-1 activity only if UBF was added to the extract prior to the immunodepletion reaction. Furthermore, SL-1 activity could be recovered in the immunoprecipitate. Interestingly, these immunoprecipitates did not contain RNA polymerase I, as a monospecific antibody to the 194-kDa subunit of RNA polymerase I failed to detect that subunit in the immunoprecipitates. Treatment of N1S1 cell extracts with the anti-UBF antibody depleted the extracts of SL-1 activity but not TFIIIB activity, suggesting that the binding of UBF to SL-1 is specific and not solely mediated by an interaction between UBF and TATA-binding protein, which is also a component of TFIIIB. These data provide evidence that UBF and SL-1 interact.

Definition of a T-cell receptor beta gene core enhancer of V(D)J recombination by transgenic mapping.

V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activating cis-recombination events. A striking example is the T-cell receptor beta-chain (TCRbeta) gene enhancer (Ebeta), which in the mouse consists of at least seven nuclear factor binding motifs (betaE1 to betaE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Ebeta required for recombination enhancer activity. The Ebeta core is comprised of a limited set of motifs (betaE3 and betaE4) and an additional previously uncharacterized 20-bp sequence 3' of the betaE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the betaE1 through betaE4 motifs but not the 3' sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Ebeta recombinational activity. Our results emphasize the multifunctionality of the TCRbeta enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.

Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system.

Traditional models for transcription initiation by RNA polymerase I include a stepwise assembly of basic transcription factors/regulatory proteins on the core promoter to form a preinitiation complex. In contrast, we have identified a preassembled RNA polymerase I (RPI) complex that contains all the factors necessary and sufficient to initiate transcription from the rDNA promoter in vitro. The purified RPI holoenzyme contains the RPI homolog of TFIID, SL-1 and the rDNA transcription terminator factor (TTF-1), but lacks UBF, an activator of rDNA transcription. Certain components of the DNA repair/replication system, including Ku70/80, DNA topoisomerase I and PCNA, are also associated with the RPI complex. We have found that the holo-enzyme supported specific transcription and that specific transcription was stimulated by the RPI transcription activator UBF. These results support the hypothesis that a fraction of the RPI exists as a preassembled, transcriptionally competent complex that is readily recruited to the rDNA promoter, i.e. as a holoenzyme, and provide important new insights into the mechanisms governing initiation by RPI.

Telomere shortening: a new prognostic factor for cardiovascular disease post-radiation exposure.

Telomere length has been proposed as a marker of mitotic cell age and as a general index of human organism aging. Telomere shortening in peripheral blood lymphocytes has been linked to cardiovascular-related morbidity and mortality. The authors investigated the potential correlation of conventional risk factors, radiation dose and telomere shortening with the development of coronary artery disease (CAD) following radiation therapy in a large cohort of Hodgkin lymphoma (HL) patients. Multivariate analysis demonstrated that hypertension and telomere length were the only independent risk factors. This is the first study in a large cohort of patients that demonstrates significant telomere shortening in patients treated by radiation therapy who developed cardiovascular disease. Telomere length appears to be an independent prognostic factor that could help determine patients at high risk of developing CAD after exposure in order to implement early detection and prevention.

Realising the European network of biodosimetry: RENEB-status quo.

Creating a sustainable network in biological and retrospective dosimetry that involves a large number of experienced laboratories throughout the European Union (EU) will significantly improve the accident and emergency response capabilities in case of a large-scale radiological emergency. A well-organised cooperative action involving EU laboratories will offer the best chance for fast and trustworthy dose assessments that are urgently needed in an emergency situation. To this end, the EC supports the establishment of a European network in biological dosimetry (RENEB). The RENEB project started in January 2012 involving cooperation of 23 organisations from 16 European countries. The purpose of RENEB is to increase the biodosimetry capacities in case of large-scale radiological emergency scenarios. The progress of the project since its inception is presented, comprising the consolidation process of the network with its operational platform, intercomparison exercises, training activities, proceedings in quality assurance and horizon scanning for new methods and partners. Additionally, the benefit of the network for the radiation research community as a whole is addressed.

[Sex ratio in the spectrum of chronobiopathology]. / Sexualproportionen im Spektrum der Chronobiopathologie.

With regard to the deficiency of representative data on the different course of the ischaemic heart disease depending upon sex a total statistics (death data) of the GDR for 5 years was elaborated and compared with the total mortality since 1903 to 1958 of the area of the former German Reich, the GDR and the FRG with still-born and deceased babies. Here conspicuous synchronous courses depending upon the season were the result. The female sex quite generally predominates the well-known course of the season of all individual mortality data, whereas the males are somewhat more endangered during the summer months. The differences established between the sexes proved in most cases as significant, in larger groups as highly significant. Moreover, the statistical reference guarantees an up to now not proven representativeness . The result of the distribution characteristics within the 10 categories examined renders possible cautious conclusions concerning the causal connection of the process.

Expression of phospholipase C isozymes by murine B lymphocytes.

Cross-linking of membrane (m) Ig, the B cell receptor for Ag, activates protein tyrosine phosphorylation and hydrolysis of phosphotidylinositol 4,5-bisphosphate. The latter signal transduction pathway is an important mediator of antigen receptor engagement. The initial event in this pathway is the activation of phospholipase C (PLC). The identity of the isozyme of PLC used in B cells and the mechanism by which it becomes activated are currently unknown. The cDNA encoding five different isozymes have been cloned. As a first step in identifying the isozyme of PLC that is coupled to mIgM, murine cDNA fragments for the five cloned PLC isozymes were generated by the polymerase chain reaction (PCR), cloned, and used to screen a panel of B cell lines representing different stages of development for PLC mRNA expression. All the B cell lines tested expressed high levels of PLC alpha and PLC gamma 2 mRNA, whereas PLC beta and PLC delta mRNA expression were undetectable by both Northern blot and PCR analysis. PLC gamma 1 had a more complicated pattern of mRNA expression. PLC gamma 1 mRNA expression was lower than that observed for PLC alpha or PLC gamma 2 mRNA and varied widely among different cell lines. The pattern of PLC gamma 1 mRNA expression did not correlate with the developmental stage of the cell lines. The pattern of PLC gamma 1 protein expression in the panel of B cell lines correlated with the pattern of PLC gamma 1 mRNA expression. PLC gamma 1 expression was very low in several B cell lines, despite the fact that these cell lines show mIgM-stimulatable PLC activity. The variable and in some cases very low expression of PLC gamma 1 suggests that it may not be the form of PLC that is activated by mIgM. In contrast, PLC alpha and PLC gamma 2 were abundantly expressed in all B cell lines tested. This observation is consistent with the possibility that PLC alpha or PLC gamma 2 is activated by mIgM.

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.

[Holiday effect in myocardial infarct]. / Festtagseffekt beim Herzinfarkt

Aimed measures of the organisation of the combat against infarction demand also the observation of temporary frequencies. On the basis of the evaluation of certificates of death of the month December of the years 1969 to 1973 of the GDR with differentiation according to so-called prehospital dead (persons who died at home and on the way to the hospital) and patients who died in the hospital with high significance an unwarrantedly high prehospital mortality during the period from Christmas to the end of the year (25th to 31st December) was established compared with the preceding week (18th to 24th December). Since in contrast to this the hospital cases and the cases "on the way" do not show any significant differences main tasks for the beginning of improvements concerning health policy may be deduced, all the more since the so-called holiday effect, expressed by a high home/clinic-relation of patients who died of myocardial infarction, could be restricted to 6 counties of the GDR on account of the analysis of further localities. From the results the tendency of a retrogression of the holiday effect is to be read off in the course of years. In the discussion an explantation of this peculiarity is attempted, and practicable conclusions for the removal and thus for the improvement the infarct situation are formulated.

[Socio-biorhythm of heart-infarct mortality--the subcycle of weekdays]. / Sozio-Biorhythmus der Herzinfarktsterblichkeit--Subzyklus der Wochentage.

Examinations of all persons who suddenly on the way died of myocardial infarction who were 70 years old and younger (so-called U-cases) in the GDR from 1970 to 1974 on the basis of certificates of death resulted in a statistically significant different distribution of persons who died on the way to the physician or in the waiting-room (so-called cases U3) compared with persons who suddenly died during transport (so-called cases U4) in 6 town-counties. From the results regional-specific causes of organizational kind in the outpatient public health and at the level of population could be derived as well as conclusions could be made for the secondary prevention by decrease of the socially determined subcycles.

[Effectiveness of various forms of therapy in chronic cardiovascular diseases--a vector analysis of the transition probabilities of degrees of severity]. / Effektivität differenter Therapieformen bei chronischen Herz-Kreislauf-Krankheiten--eine Vektorenanalyse der Ubergangswahrscheinlichkeiten von Schweregraden.

The present findings result from a check of probationers representative for approx. 280,000 inhabitants of defined territories, who had been designated as suspects of heart and vessel disease on grounds of X-ray-morphological criterions (classification by Richter). Those about 3,000 suspects, subdivided into 3 comparable patient groups A, B and C, underwent different regimes of treatment of outpatient medical practice after standardized and noninvasive diagnostics in a follow-up during 5 years and had been valued by means of a problem-specific grading. The comparative analysis about the distribution of severe degrees concerning hypertension and coronary heart disease after the conclusion of the intervention showed significant differences concerning the results of treatment to the credit of the patient group A (treated by specialists) contrary to the patient group B (treated by family doctors) and patient group C (principle of announcing the patients themselves). Also the patient group B showed significantly better results of therapy compared with the patient group C. In addition to the concluding rating the estimation of yearly transition of severe degrees gave an insight into the therapeutical decision of the person who looks after as well as the different distribution of severe degrees of special heart diseases in dependence on the starting severe degree in the special period of intervention conditioned on the therapy. The results gain exceptional importance for practice on the grounds of methodics of the study-automatable classification of dv-thorax-X-ray pictures, problem-specific grading of noninvasive, simple parameters, variants of therapy in dependence on the graduated system of medical care.

[Value and rank of diagnostic laboratory parameters in cardiovascular diseases. Results of multivariant and discriminant analyses of the Berlin EBMO-Cor population study]. / Aussagefähigkeit und Rangfolge labordiagnostischer Parameter bei Herz-Kreislauf-Krankheiten. Ergebnisse multivariater Varianz- und Diskriminanzanalysen aus der bevölkerungsrepräsentativen Studie "EBMO-Cor"-Berlin.

Based on present clinical-chemical parameters a representative population were studied; importance and order of rank of 10 data chosen from a total data pool are determined by means of multivariant and discriminant analysis. As a result the validity of characters with and without apriori probabilities for 5 pairs of classes (persons suffering from a heart disease in comparison to healthy persons divided in sexes and overall, as well as examination of separate sexes for healthy persons and those suffering from a heart disease) are examined (alpha less than or equal to 0.05). This calculation tested the importance of the parameters hemoglobin and creatinine in being different in males and females. To diagnose persons suffering from a heart disease (X-ray-morphologically defined suspects of heart and vessel diseases) from healthy persons, the optimized number of characteristics were determined in cholesterol, erythrocyte sedimentation rate, ASAT, blood glucose (independent of sex). By means of these results it is possible to identify persons suffering from a heart disease from healthy persons and to call for illustrative laboratory examinations using already mentioned parameters and the function of discriminance W = p sigma i = 1 ai X Xi. The determination of sensitivity and specification yielded a value of up to 98 per cent resp. 94 per cent depending on whether with or without apriori probabilities. This enables use to be modified to suit different purposes.

[Correlation of pathologic laboratory values in patients with cardiovascular diseases--consequences for diagnosis in general practice]. / Die Korrelation pathologischer Laborwerte bei Herz-Kreislauf-Kranken--Konsequenzen für die Diagnostik in der Praxis.

Coincident pathological parameters were selected from 24 laboratory-diagnostic parameters of a second stage of diagnostics after x-rays screening concerning heart and vessel diseases tested by a check representative constellation in the shape of relative frequency was determined. For the parameters cholesterol, uric acid and glucose, belonging to the metabolic syndrome, it was possible to demonstrate relations to the erythrocyte sedimentation rate. The transaminases ASAT and ALAT especially showed correlations of pathological values among one another. There were found out one-sided relations for instance concerning the proportion of transaminases, thymol turbidity test as well as creatinine to the erythrocyte sedimentation rate. The connections as has been proved appeared in female cardiac patients in a more distinct way. The results, which were interpreted in the context of a further mathematic-statistical analysis, allow the conclusions for an efficient indicational application of clinical chemical research methods in chronic heart and vessel diseases in practice.

[Comparative biochemical parameters in deceased and living patients in a follow-up intervention study of cardiovascular patients]. / Vergleiche biochemischer Parameter bei Verstorbenen und Nichtverstorbenen einer follow-up-Interventionsstudie von Herz-Kreislauf-Kranken.

After the laboratory-diagnostical spectrum had been analysed by means of a representative check of probationer with a state suspected of heart and vessel diseases on the screen of the X-ray mass examination in longitudinal section and cross-section with derived conclusions the results had been checked by means of comparison with the dead and not dead of this population in the follow up. The parameter sedimentation rate of erythrocyte, blood sugar, the enzymes "ASAT" and "ALAT" and total cholesterol with stronger pronounced proneness could substantiate the causal connection to this population of persons suffering from a heart disease as well. Hence followed derived relations to the severe degree of heart and vessel disease and beside this to the prognostical estimation of the disease for the parameter uric acid, hemoglobin/hematocrit and creatinine in the serum. The number of leucocytes, the serum protein, the separation of fractions of serum protein and lability test of serum by electrophoresis did not result in any findings, which allowed any connection with heart and vessel diseases, also concerning the persons who died in the course of the study, so that the demand for a differentiated, well-aimed differential-diagnostical use of these expensive research methods also for this extreme situation is substantiated once more. The results are discussed in comparison as well as in connection with the results found out of the total check and in dependence of age and sexes.

[Methodology and organization of a laboratory diagnostic study program within the scope of epidemiologic cardiovascular studies exemplified by the Berlin EMBO-Cor research project]. / Methodik und Organisation eines labordiagnostischen Untersuchungsprogramms im Rahmen epidemiologischer Herz-Kreislauf-Studien am Beispiel des Forschungsprojektes EBMO-Cor Berlin.

With reference to the high prevalence of heart and vessel disease and the necessity of its early detection procedure and consideration concerning a laboratory-diagnostic program within the framework of the follow-up examination after x-ray screening (research project EBMO-Cor Berlin) in special consideration of efficient methods within the territorial Public Health are depicted and also conclusions for similar epidemiological examinations and for practice are formulated.