Intercomparison on the measurement of the thoron exhalation rate from building materials.

Thoron (220Rn) exhalation from building materials has become increasingly recognized as a potential source for radiation exposure in dwellings. However, contrary to radon (222Rn), limited information on thoron exposure is available. As a result no harmonized test procedures for determining thoron exhalation from building materials are available at present. This study is a first interlaboratory comparison of different test methods to determine the thoron exhalation and a pre-step to a harmonized standard. The purpose of this study is to compare the experimental findings from a set of three building materials that are tested, and to identify future challenges in the development of a harmonized standard.

Preparation and characterisation of ceramic-based thoron sources for thoron calibration chamber.

The aim of this study is to explore the correlations between the properties of the source's material and the thoron flux produced. This means a complex procedure that involves morphological characterisation (the determination of specific surface area and pore size distribution) and thoron emanation and exhalation measurements as well. In this work, the preparation of 27 thoron sources has been carried out. Three types of ceramics with different morphological properties were used as a matrix material with three different thorium contents. Spheres were formed from the dollop, and they were fired at different temperatures (200, 600 and 900°C). The phase analysis of the samples was performed by powder X-ray diffraction. The pore size distribution was determined by mercury penetration. The thoron emanation was measured using an accumulation chamber; the measured thoron emanation coefficients were from 0.34 ± 0.03 to 7.69 ± 0.13 %. Based on the results, the preparation parameters of the thoron source optimised for the calibration procedure have been given.

Calibration of CR-39-based thoron progeny device.

Radon isotopes and their progenies have proven significant role in respiratory tumour formation. In most cases, the radiological effect of one of the radon isotopes (thoron) and its progenies has been neglected together with its measurement technique; however, latest surveys proved that thoron's existence is expectable in flats and in workplace in Europe. Detectors based on different track detector measurement technologies have recently spread for measuring thoron progenies; however, the calibration is not yet completely elaborated. This study deals with the calibration of the track detector measurement method suitable for measuring thoron progenies using different devices with measurement techniques capable of measuring several progenies (Pylon AB5 and WLx, Sarad EQF 3220). The calibration factor values related to the thoron progeny monitors, the measurement uncertainty, reproducibility and other parameters were found using the calibration chamber. In the future, the effects of the different parameters (aerosol distribution, etc.) will be determined.

Preliminary results of indoor radon survey in V4 countries.

The measurements of radon activity concentration carried out in residential houses of V4 countries (Hungary, Poland and Slovakia) show that radon levels in these countries considerably exceed the world average. Therefore, the new radon data and statistical analysis are required from these four countries. Each partner chose a region in their own country, where radon concentration in residential buildings was expected to be higher. The results of the survey carried out in the period from March 2012 to May 2012 show that radon concentrations are <200 Bq m(-3) in ∼87% of cases. However, dwellings with radon concentration ∼800 Bq m(-3) were found in Poland and Slovakia. It was also found that the distribution of radon frequency follows that of houses according to the year of their construction.

Preliminary results from an indoor radon thoron survey in Hungary.

More than half of the radiation dose of natural origin comes from radon. However, according to some surveys in certain cases, the radiation dose originating from thoron may be considerable. Among the factors disturbing the measurement of radon, the presence of thoron may also influence the measured radon value, making the estimated radiation exposure imprecise. Thoron has previously been surveyed, mainly in Asia; however, recent surveys for some European locations have found that significant thoron concentrations also need to be considered. In this survey, several types of commercially available SSNTDs (solid-state nuclear track detectors) capable of measuring both radon and thoron were placed at the same time in 73 houses and 7 workplaces in Hungary with 3-month exposition periods. In order to measure thoron, the distance of the detector sets was fixed as 15-20 cm from the walls. The radon concentration was measured with five types of SSNTDs: NRPB, NRPB SSI, Raduet, DTPS and DRPS. The first four types had relatively good accordance (within ± 10 %), but the results of the DRPS detectors were considerably lower when compared with other detectors for radon concentrations over 100 Bq m(-3). The thoron averages were provided by two different types of detectors: Raduet and DTPS. The difference between their average results was more than 30 % and was six times the maximum values. Therefore, the thoron measurement results were judged to be erroneous, and their measurement protocol should be clearly established for future work.

Rapid 'glycaemic swings' induce nitrosative stress, activate poly(ADP-ribose) polymerase and impair endothelial function in a rat model of diabetes mellitus.

AIM/HYPOTHESIS: Postpandrial hyperglycaemia is a significant risk factor for the development of macrovascular diseases. There is no clear agreement in the field whether these alterations result from hyperglycaemic episodes or from exaggerated alterations ('glycaemic swings') in blood glucose. We compared the effect of stable high glucose with a model of poorly maintained insulin-controlled diabetes (on average lower glucose, but with large glycaemic swings) on the development of endothelial dysfunction in rats. METHODS: Intermediate- or long-acting insulin was used to reduce mean blood glucose levels. One group of animals had stable low glucose levels, while animals in the other group exhibited rapid changes ('swings') in their blood glucose concentration. Acetylcholine-induced endothelium-dependent vascular relaxation of the thoracic aorta was measured. Immunohistochemistry, western blot analysis and flow cytometry were used to determine nitrotyrosine formation and poly(ADP-ribose) accumulation in the aorta, in circulating leucocytes and in bone marrow cells. RESULTS: Steady normalisation of blood glucose levels (a model of well-controlled diabetes) protected against the development of endothelial dysfunction, poly(ADP-ribose) polymerase (PARP) activation and nitrotyrosine production. However, impairment of endothelium-dependent relaxation was found in the animals undergoing glycaemic swings, even though the fructosamine levels in these animals were lower than in the untreated diabetic rats. This was associated with elevated PARP activation in the aorta and in bone marrow cells that was similar to or even more pronounced than that seen in the untreated diabetic animals. CONCLUSIONS/INTERPRETATION: Large glycaemic swings exert deleterious cardiovascular effects in diabetes mellitus, in part via enhanced activation of the PARP pathway.

Comparison of primers for the detection of Salmonella enterica serovars using real-time PCR.

AIMS: To evaluate the specificity and sensitivity of PCR primers for the detection of Salmonella enterica in a real-time PCR assay using pure cultures. METHODS AND RESULTS: Unenriched whole cells in sterile water were used as template for each PCR. SYBR Green dye was used for the nonspecific detection of dsDNA. The real-time PCR detection limits of five previously published primer sets used in conventional PCR applications were not below 3 x 10(3) CFU per reaction (rxn). A new primer set, Sen, was designed, which detected Salm. enterica Newport down to 6 CFU rxn(-1) in one case, and gave an average detection limit of 35 CFU rxn(-1) over three separate runs. CONCLUSIONS: Primers originally designed for end-point PCR did not have adequate specificity or sensitivity compared with those specifically designed for real-time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study emphasizes the importance of evaluating real-time PCR primer sets in pure cultures prior to testing in field samples. This study will benefit other researchers in selecting an appropriate primer set for real-time PCR detection of Salm. enterica.

Recovery versus death of "dark" (compacted) neurons in non-impaired parenchymal environment: light and electron microscopic observations.

The formation of massively shrunken, hyperbasophilic, hyperargyrophilic and hyper-electron-dense but not apoptotic ("dark") neurons was initiated in rat brains by means of an electric-shock and two mechanical-injury paradigms that do not cause considerable parenchymal damage in the areas investigated. The rats were killed by perfusion fixation either immediately after these instantaneous initiating insults or after a survival period ranging from 40 min to 6 days. The formation of "dark" neurons was complete in less than a few minutes. In the somatodendritic domain of each "dark" neuron, all ultrastructural elements were remarkably preserved during the acute stage, apart from a dramatic reduction of the distances between them. This ultrastructural compaction was accompanied by a marked shift of cell fluid through seemingly intact plasma membrane, mainly into surrounding astrocytic elements. The majority of the "dark" neurons regained their normal morphology and staining properties (recovery) in 4 h. Thereafter, only solitary mitochondrion-derived membranous whorls in the cytoplasm reminded of a previous morphological disturbance. The dead "dark" neurons fell apart into membrane-bound fragments that retained their sharp outlines and compacted interior even after being engulfed by astrocytes or microglial cells. The latter sequence of morphological changes can not be harmonized with the prevailing assumption, according to which "dark" neurons die through the necrotic pathway. The fate of the "dark" neurons appeared to depend on the presence or absence of serious post-insult pathophysiological circumstances in their surroundings.

Inhibition of histone deacetylase activity enhances Fas receptor-mediated apoptosis in leukemic lymphoblasts.

We recently reported that butyrate, an inhibitor of histone deacetylases, is capable of inducing Fas-independent apoptosis in the acute lymphoblastic leukemia cell line CCRF-CEM. Here we demonstrate that butyrate enhances Fas-induced apoptosis in this cell line. The application of different histone deacetylase inhibitors revealed that tetra-acetylated histone H4 is associated with the amplifying effect of butyrate on Fas-induced cell death. FasL, Fas, FADD, RIP, caspase-8, caspase-3, Bid, FLIP(S+L), FLASH and FAP-1, proteins known to act within the Fas-apoptosis cascade, showed no changes in their expression levels in cells treated with butyrate compared with untreated cells. Analyses of Fas-oligomerization and Western blotting as well as enzyme activity assays of caspase-2, caspase-3 and caspase-8 suggest that butyrate enhances Fas-induced apoptosis downstream of Fas but upstream of caspase-8 activation. In immunoprecipitation experiments a 37 kD butyrate-regulated protein was detected which specifically interacts with caspase-8.

Resveratrol causes arrest in the S-phase prior to Fas-independent apoptosis in CEM-C7H2 acute leukemia cells.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene), in the concentration range of 20 microM and above, induced arrest in the S-phase and apoptosis in the T cell-derived T-ALL lymphocytic leukemia cell line CEM-C7H2 which is deficient in functional p53 and p16. Expression of transgenic p16/INK4A, which causes arrest in G0/G1, markedly reduced the percentage of apoptotic cells. Antagonist antibodies to Fas or FasL, or constitutive expression of crmA did not diminish the extent of resveratrol-induced apoptosis. Furthermore, a caspase-8-negative, Fas-resistant Jurkat cell line was sensitive to resveratrol-induced apoptosis which could be strongly inhibited in the Jurkat as well as in the CEM cell line by z-VAD-fmk and z-IETD-fmk. The almost complete inhibition by z-IETD-fmk and the lack of inhibition by crmA suggested caspase-6 to be the essential initiator caspase. Western blots revealed the massive conversion of procaspase-6 to its active form, while caspase-3 and caspase-2 were proteolytically activated to a much lesser extent.

Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts.

The histone deacetylase inhibitor and potential anti-cancer drug sodium butyrate is a general inducer of growth arrest, differentiation, and in certain cell types, apoptosis. In human CCRF-CEM, acute T lymphoblastic leukemia cells, butyrate, and other histone deacetylase inhibitors caused G2/M cell cycle arrest as well as apoptotic cell death. Forced G0/G1 arrest by tetracycline-regulated expression of transgenic p16/INK4A protected the cells from butyrate-induced cell death without affecting the extent of histone hyperacetylation, suggesting that the latter may be necessary, but not sufficient, for cell death induction. Nuclear apoptosis, but not G2/M arrest, was delayed but not prevented by the tripeptide broad-range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD) and, to a lesser extent, by the tetrapeptide 'effector caspase' inhibitors benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Glu-Ile-Asp.fluoromethyl-ketone (VEID); however, the viral protein inhibitor of 'inducer caspases', crmA, had no effect. Bcl-2 overexpression partially protected stably transfected CCRF-CEM sublines from butyrate-induced apoptosis, but showed no effect on butyrate-induced growth inhibition, further distinguishing these two butyrate effects. c-myc, constitutively expressed in CCRF-CEM cells, was down-regulated by butyrate, but this was not causative for cell death. On the contrary, tetracycline-induced transgenic c-myc sensitized stably transfected CCRF-CEM derivatives to butyrate-induced cell death.

Interaction between dexamethasone and butyrate in apoptosis induction: non-additive in thymocytes and synergistic in a T cell-derived leukemia cell line.

In thymocytes butyrate and trichostatin A are unable to augment dexamethasone-induced apoptosis. In cultured rat thymocytes the extent of apoptosis induced by dexamethasone alone did not increase by addition of 0.1 - 10 mM butyrate. Even more pronounced was the non-additive interrelationship between dexamethasone and trichostatin A, as trichostatin A-induced apoptosis was not only blocked by the presence of dexamethasone but dexamethasone-induced apoptosis was also partially inhibited in the presence of 0.1 - 0.5 microM trichostatin A. The fact that the non-additive relationship with dexamethasone for apoptosis induction was observed with both histone deacetylase inhibitors suggests that in thymocytes this phenomenon is related to histone acetylation. In contrast to this, in the human T cell-derived leukemia cell line CEM-C7H2, dexamethasone did not block butyrate- or trichostatin A-induced apoptosis; moreover, butyrate, in the concentration range of 0.1 - 1 mM, had a marked synergistic effect on dexamethasone-induced apoptosis. This synergism, however, was not mimicked by trichostatin A, indicating that the effect is not related to histone acetylation but rather due to a pleiotropic effect of butyrate. Furthermore, in CEM-C7H2 cells, at higher concentrations of butyrate (5 - 10 mM) or trichostatin A (0.4 - 0.8 microM), there was a minor but reproducible antagonistic effect of dexamethasone on apoptosis induced by each of the two histone deacetylase inhibitors, suggesting that this antagonistic effect too, is related to histone hyperacetylation.

Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins.

Basic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two-dimensional (2-D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4-10 and 6-10 using a previously published protocol (Görg et al., Electrophoresis 1988, 9, 531-546). In the present study we have extended the pH gradient to pH 12 with IPGs 8-12, 9-12 and 10-12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of isopropanol with and without methylcellulose to the IPG rehydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2-D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady-state conditions. Due to the selectivity of isoelectric focusing in IPG 9-12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone extraction can be omitted.

Butyrate, aspirin and colorectal cancer.

In vitro, for animal cells generally, butyrate at millimolar concentrations is an inhibitor of growth. In vivo, however, colonocytes are able to grow in the environment of about 20 mM butyrate produced by bacterial fermentation on the luminal side of the colonic epithelium. An in vivo increase of the butyrate supply results in growth stimulation of cells in the colonic crypts. This discrepancy, namely, that in cell cultures butyrate is an inhibitor of growth, whereas in vivo it has a trophic effect, is the so called in vivo paradox of butyrate. In the present review it is pointed out that butyrate is an inhibitor of histone deacetylases and there is sufficient evidence for hyperacetylation being the mechanism of the in vitro growth-inhibiting effect of butyrate. As within animal cells hyperacetylation has to occur at a certain butyrate concentration (1-10 mM), it is postulated that the in vivo lack of inhibition and 'paradoxical' stimulation of growth is a result of a low intracellular steady state concentration of butyrate in the lower layers of the crypt in spite of the much higher butyrate concentration on the luminal side. As butyrate is the preferential source of energy for colonocytes, the in vivo trophic effect is not paradoxical, when in spite of an increase of the butyrate concentration in stool, the intracellular butyrate concentration of intestinal epithelial cells still remains below the inhibiting level. For mature non-dividing colonocytes which are programmed for apoptosis, there is no difference between the observations made in vitro or in vivo. Furthermore, recent developments are discussed which suggest that cyclo-oxygenase-2 may play an essential role in colonic carcinogenesis. Cyclo-oxygenase-2 is found to be expressed in most colorectal carcinomas, but not in normal non-transformed intestinal epithelial cells (DeWitt and Smith, 1995). Cyclo-oxygenase-2 overexpression makes intestinal epithelial cells resistant to butyrate-induced apoptosis (Tsujii and DuBois, 1995). This escape from butyrate-induced apoptosis appears to be an essential prerequisite for the development of colorectal cancer and suggests a functional role of butyrate in growth, differentiation and programmed cell death of colonic epithelial cells.