Indoor radon risk mapping of the Canary Islands using a methodology for volcanic islands combining geological information and terrestrial gamma radiation data.

Within the framework of the recent approval of the National Plan Against Radon by the Council of Ministers of the Spanish Government, one of its five axes focuses on the delimitation of priority action areas. In line with this objective, this paper presents the indoor radon risk maps of the Canary Islands. Due to the volcanic origin of the Canary Islands, there is a great deal of geological heterogeneity in the soils on which buildings settle, making it very difficult to delimit radon-risk areas in the process of creating maps. Following a methodology developed in previous works for a study area formed of a set of representative municipalities, this paper presents radon risk maps of the Canary Islands based on lithostratigraphic information and high-resolution terrestrial gamma radiation maps. The goodness of fit of these maps is verified based on a statistical analysis of indoor radon concentration measurements carried out at representative building enclosures. In order to analyse the level of risk to the population, these maps were combined with built up areas (urban fabric) maps and estimations of the annual effective doses due to radon was obtained by applying a dosimetric model. This methodology improves the capability to delimit indoor radon risk areas, with a greater margin of safety. In this respect, it is estimated that areas classified as low risk have indoor radon concentrations 41 % below the current reference level of 300 Bq/m3 established by national regulations in compliance with the precepts laid down in the European EURATOM Directive.

Forgotten giants: Robust climate signal in pollarded trees.

Tree ring records are among the most valuable resources to create high-resolution climate reconstructions. Most climate reconstructions are based on old trees growing in inaccessible mountainous areas with low human activity. Therefore, reconstruction of climate conditions in lowlands is usually based on data from distant mountains. Albeit old trees can be common in humanized areas, they are not used for climate reconstructions. Pollarding was a common traditional management in Europe that enabled trees to maintain great vitality for periods exceeding the longevity of unmanaged trees. We evaluate the potential of pollarded deciduous oaks to record past climate signal. We sampled four pollarded woodlands in Central Spain under continental Mediterranean climate. We hypothesized that pollarded trees have a strong response to water availability during current period without pollarding management, but also in the period under traditional management if pruning was asynchronous among trees. Moreover, we hypothesized that if climate is a regional driver of oak secondary growth, chronologies from different woodlands will be correlated. Pollard oaks age exceeded 500 years with a strong response to Standardized Precipitation-Evapotranspiration Index (SPEI) from 9 to 11 months. Climate signal was exceptionally high in three of the sites (r2 = 0.443-0.655) during low management period (1962-2022). The largest fraction of this climate signal (≈70 %) could be retrieved during the traditional management period (1902-1961) in the three sites where pollarding was asynchronous. Chronologies were significantly correlated since the 19th century for all the studied period, highlighting a shared climate forcing. We identified critical points to optimize pollard tree sampling schema. Our results show the enormous potential of pollarded woodlands to reconstruct hydroclimate conditions in the Mediterranean with a fine spatial grain. Studying pollarded trees is an urgent task, since the temporal window to retrieve the valuable information in pollarded trees is closing as these giants collapse and their wood rots.

Multiparametric analysis for the determination of radon potential areas in buildings on different soils of volcanic origin.

The transposition of the European EURATOM directive into the regulations of the different member states of the European Union involved governments making great efforts to define priority action maps against indoor radon exposure in buildings over a short time period. In Spain, the Technical Building Code established 300 Bq/m3 as a reference level and set up a classification of municipalities in which remediation measures should be adopted for radon exposure in buildings. Oceanic volcanic islands, such as the Canary Islands, present high geological heterogeneity in a small space due to their volcanic origin. This variability poses a challenge to the elaboration of radiological risk maps, which makes it necessary to have a high density of data to collect local variations. This paper presents a methodology to obtain accurate radon risk maps based on geological criteria and terrestrial gamma radiation. The predictive efficiency of these maps is statistically verified using indoor radon concentration data measured in buildings. Other radiological variables, which are commonly used as criteria for radon risk prediction found in the literature, were also applied, such as the geogenic radon potential and the activity concentration of natural radioisotopes in soils. The higher resolution of the maps obtained allows for a more detailed classification of radon risk zones in the study area than the current risk maps published in the Spanish building regulations.

Effect of metabolic control on recurrent major adverse cardiovascular events and cardiovascular mortality in patients with premature coronary artery disease: Results of the Genetics of Atherosclerotic Disease study.

BACKGROUND AND AIMS: Coronary artery disease (CAD) is the leading cause of death around the world, and its rate of presentation is increasing at young ages. Despite the evidence that secondary prevention in CAD reduces the risk of recurrent major adverse cardiovascular events (MACE), no studies have analyzed the composite control of blood pressure, lipids, and glucose control in premature CAD. METHODS AND RESULTS: This was a real-world prospective cohort study of patients with premature CAD. The composite control in blood pressure <140/80 mmHg, LDL-C <70 mg/dL, non-HDL-C <100 mg/dL, and Hemoglobin A1c <8% was considered as metabolic control. The primary endpoint was the occurrence of non-fatal and fatal MACE. The data included 1042 patients with premature CAD. The mean age of the patients was 54.1 ± 8.1 years, 18.5% were women, and had a median follow-up of 59.1 ± 11.8 months. Of them, 7% had non-fatal MACE, and 4% had a fatal MACE. Overall, 21.3% achieved metabolic control, and 3.0% did not achieve any target. Cox regression analysis showed that percutaneous coronary intervention (Hazzard ratio = 1.883 [95% CI, 1.131-3.136]), C-reactive protein (1.046 [1.020-1.073]), blood pressure >140/90 mmHg (2.686 [1.506-4.791]), fibrates (2.032 [1.160-3.562]), calcium channel blockers (2.082 [1.158-3.744]) had greater risk to present a recurrent non-fatal MACE; whereas familial history of premature CAD (2.419 [1.240-4.721]), heart failure (2.139 [1.032-4.433]), LDL-C >70 mg/dL (4.594 [1.401-15.069]), and diuretics (3.328 [1.677-6.605]) were associated with cardiovascular mortality. CONCLUSIONS: The composite goal achievement in lipids, blood pressure and glucose, reduced the risk for recurrent MACE in 80%.

Frequency, profile, and outcomes of patients with acute heart failure transferred directly to home hospitalization from emergency departments.

OBJECTIVE: To describe the frequency, clinical characteristics and outcomes of patients with acute heart failure (AHF) transferred directly from emergency departments to home hospitalisation (HH) and to compare them with those hospitalised in internal medicine (IM) or short-stay units (SSU). METHOD: We included patients with AHF transferred to HH by hospitals that considered this option during the Epidemiology of Acute Heart Failure in Spanish Emergency Departments (EAHFE) 4-5-6 Registries and compared them with patients admitted to IM or SSU in these centres. We compared the adjusted all-cause mortality at 1 year and adverse events 30 days after discharge. RESULTS: The study included 1473 patients (HH/IM/SSU:68/979/384). The HH rate was 4.7% (95% CI 3.8-6.0%). The patients in HH had few differences compared with those hospitalised in IM and SSUs. The HH mortality was 1.5%, and the HH median stay was 7.5 days (IQR, 4.5-12), similar to that of IM (median stay, 8 days; IQR, 5-13; p = .106) and longer than that of SSU (median stay, 4 days; IQR, 3-7; p < .001). The all-cause mortality at 1 year for HH did not differ from that of IM (HR, 0.91; 95% CI 0.73-1.14) or SSU (HR, 0.77; 95% CI 0.46-1.27); however, the emergency department readmission rate during the 30 days postdischarge was lower than that of IM (HR, 0.50; 95% CI 0.25-0.97) and SSU (HR, 0.37; 95% CI 0.19-0.74). There were no differences in the need for new hospitalisations or in the 30-day mortality rate. CONCLUSIONS: Direct transfer from the emergency department to HH is infrequent despite being a safe option for a certain patient profile with AHF.

Methodology for determination of radon prone areas combining the definition of a representative building enclosure and measurements of terrestrial gamma radiation.

The recommendations of the European Atomic Energy Community (EURATOM) have recently been incorporated into Spanish regulations in the Basic Document of Health Standards of the Technical Building Code (CTE), section HS6, on protection against radon exposure. This further accentuates the need to delimit radon prone areas as a strategy to address measures which minimise the effects of this gas on the population. In this research, measurements of terrestrial gamma radiation and indoor radon of dwellings have been carried out in the same location to delimit these risk areas. A new methodology has been developed including a definition of a Representative Building Enclosure (RBE) and it is proposed a Building Storey Index (IBS) which allows normalizing measurements of indoor radon activity concentration taken in different levels from the ground to the RBE. The results show the need to consider the type of contact that exists between the building and the ground as a determining factor of radon risk. Terrestrial gamma radiation is used as a proxy for radioisotopic composition of soils to characterise the indoor radon risk at different geological formation.

Frequency, profile and results of patients with acute heart failure transferred directly to home hospitalisation from emergency departments. / Frecuencia, perfil y resultados de los pacientes con insuficiencia cardiaca aguda que ingresan directamente en hospitalización a domicilio desde urgencias.

OBJECTIVE: To describe the frequency, clinical characteristics and outcomes of patients with acute heart failure (AHF) transferred directly from emergency departments to home hospitalisation (HH) and to compare them with those hospitalised in internal medicine (IM) or short-stay units (SSU). METHOD: We included patients with AHF transferred to HH by hospitals that considered this option during the Epidemiology of Acute Heart Failure in Spanish Emergency Departments (EAHFE) 4-5-6 Registries and compared them with patients admitted to IM or SSU in these centres. We compared the adjusted all-cause mortality at 1 year and adverse events 30 days after discharge. RESULTS: The study included 1473 patients (HH/IM/SSU: 68/979/384). The HH rate was 4.7% (95% CI, 3.8-6.0%). The patients in HH had few differences compared with those hospitalised in IM and SSUs. The HH mortality was 1.5%, and the HH median stay was 7.5 days (IQR, 4.5-12), similar to that of IM (median stay, 8 days; IQR, 5-13; p=.106) and longer than that of SSU (median stay, 4 days; IQR, 3-7; p<.001). The all-cause mortality at 1 year for HH did not differ from that of IM (HR, 0.91; 95% CI, 0.73-1.14) or SSU (HR, 0.77; 95% CI, 0.46-1.27); however, the emergency department readmission rate during the 30 days postdischarge was lower than that of IM (HR, 0.50; 95% CI, 0.25-0.97) and SSU (HR, 0.37; 95% CI, 0.19-0.74). There were no differences in the need for new hospitalisations or in the 30-day mortality rate. CONCLUSIONS: Direct transfer from the emergency department to HH is infrequent despite being a safe option for a certain patient profile with AHF.

Assessment of radon risk areas in the Eastern Canary Islands using soil radon gas concentration and gas permeability of soils.

The Basic Safety Standard (BSS) Directive 2013/59/EURATOM of the European Union (EU) has stated the need for member states to establish national action plans to mitigate their general population's long-term risks of exposure to radon gas. Maps of radon-prone areas provide a useful tool for the development of such plans. This paper presents the maps of radon-prone areas in the Eastern Canary Islands (Gran Canaria, Fuerteventura and Lanzarote) obtained from assessment of Geogenic Radon Potential (GRP) distribution in the territory. GRP constitutes a magnitude that is contingent on both radon activity concentration and gas permeability of soils. An extensive campaign covering all geological formations of the Eastern Canary Islands was undertaken to locally sample these parameters. Geostatistical analysis of the spatial distribution of radon concentration in soils, permeability and GRP was performed on each of the islands, and the relationship between these magnitudes and the characteristic geological formations of the volcanic islands was investigated. Areas dominated by basic volcanic and plutonic rocks (originated by both recent and ancient volcanism) exhibit relatively low levels of radon in soils, and with the exception of specific cases of very high permeability, these areas are not classified as prone to radon risk according to international criteria. Areas in which intermediate or acidic volcanic and plutonic rocks predominate are characterised by greater radon activity concentration in soils, rendering them radon-prone. Given these results, Lanzarote is classified as an island with low radon risk all over its surface; Fuerteventura presents low-medium risk; and Gran Canaria contains extensive areas in the centre and north where the risk is medium or high. This classification is consistent with the risk maps obtained by National and European agencies from indoor radon measurements conducted on these islands.

Natural radioactivity in algae arrivals on the Canary coast and dosimetry assessment.

Nowadays, the use of wild and culture harvest seaweed in food industry is a booming productive sector. In this context, a radiological characterization of five globally common seaweed species that were collected in arrival on Gran Canaria coast was carried out. The studied algae species were Cymopolia barbata, Lobophora variegata, Sargassum vulgare, Dictyota dichotoma and Haliptilon virgatum. Radionuclides analysed by alpha and gamma spectrometry were 238U, 234U, 235U, 210Po, 234Th, 226Ra, 210Pb, 228Th, 224Ra, 40K and 7Be. Activity concentrations, ratios, and concentration factors (CF) were determined for all samples collected. The CF in algae was higher for reactive-particle radionuclides (210Po, 234Th, 228Th and 210Pb) than for conservative ones (40K and the uranium isotopes). 210Po, 228Th and 234Th CF were one or two orders of magnitude higher than those recommended by the IAEA. L. variegata, C. barbata and S. vulgare showed a clear preference for 210Pb and 210Po, for uranium radioisotopes, and for 40K and 234Th, respectively. A dosimetry assessment due to seaweed ingestion showed considerable values of annual committed effective dose for H. virgatum (605 ±â€¯19 µSv/y), L. variegata (574 ±â€¯17 µSv/y) and D. dichotoma (540 ±â€¯30 µSv/y). Hence, this study suggests that an algae radiological characterization is recommended as part of the product valorising process.

The diet of a nocturnal pelagic predator, the Bulwer's petrel, across the lunar cycle.

The lunar cycle is believed to strongly influence the vertical distribution of many oceanic taxa, with implications for the foraging behaviour of nocturnal marine predators. Most studies to date testing lunar effects on foraging have focused on predator activity at-sea, with some birds and marine mammals demonstrating contrasting behavioural patterns, depending on the lunar-phase. However, to date no study has focused on how the lunar cycle might actually affect predator-prey interactions in the upper layers of the ocean. Here, we tested whether the diet of the predominantly nocturnal pelagic predator, the Bulwer's petrel (Bulweria bulwerii) would change throughout the lunar cycle, using molecular analysis to augment detection and taxonomic resolution of prey collected from stomach-contents. We found no evidence of dietary shifts in species composition or diversity, with Bulwer's petrel always consuming a wide range of mesopelagic species. Other co-variables potentially affecting light availability at-sea, such as percentage of cloud cover, did not confound our results. Moreover, many of the species found are thought not to reach the sea-surface. Our findings reveal that nocturnal predators are probably more specialized than previously assumed, irrespective of ambient-light, but also reveal deficiencies in our current understanding of species vertical distribution and predation-dynamics at-sea.

Mapping natural radioactivity of soils in the eastern Canary Islands.

The Canary Islands archipielago (Spain) comprises seven main volcanic islands and several islets that form a chain extending for around 500 km across the eastern Atlantic, between latitudes 27°N and 30°N, with its eastern edge only 100 km from the NW African coast. The administrative province of Las Palmas comprises the three eastern Canary Islands (Lanzarote, Fuerteventura and Gran Canaria). An extensive study of terrestrial gamma dose rates in surface soils has been carried out to cover the entire territory of the province (4093 km2). The average outdoor gamma dose rate in air at 1 m above ground is 73 nGyh-1 at Gran Canaria, 32 nGyh-1 at Fuerteventura, and 25 nGyh-1 at Lanzarote. To complete the radiological characterization of this volcanic area, 350 soil samples at 0-5 cm depth were collected to cover all the geologic typologies of the islands. These samples were measured using high resolution gamma spectrometry to determine the activity concentrations of 226Ra, 232Th and 40K. The average values obtained were 25.2 Bq/kg, 28.9 Bq/kg, and 384.4 Bq/kg, respectively. Maps of terrestrial gamma activity, effective dose, and activity concentrations of 226Ra, 232Th and 40K for the region have been developed through the use of geostatistical interpolation techniques. These maps are in accord with the geology of the islands.

Thyroid stimulating hormone directly modulates cardiac electrical activity.

BACKGROUND: The electrocardiogram of hypothyroid patients shows a series of abnormalities of cardiac repolarization due to a reduction of some repolarizing K(+) currents and an increase of the L-type calcium current. Experimental and clinical works call into question the unique role of T3 and T4 in these mechanisms and correlate increased serum TSH levels with the repolarization abnormalities in patients with both subclinical and overt hypothyroidism. In this context, the aim of the present study was to investigate the direct effects of TSH upon cardiac electrical properties. METHODS: The action potential recording and the ion channel subunits mRNA expression were obtained from left ventricle of adult rats. Additionally, the repolarizing K(+) currents and the L-type Ca(2+) current (ICa-L) were recorded in isolated rat adult ventricular myocytes by the patch-clamp technique. RESULTS: 24h exposure to TSH lengthened the action potential and slightly depolarized the resting membrane potential. TSH- receptor activation causes a reduction of the amplitude of Ito and IK1 currents caused by a reduction in channels expression. However, TSH had no effect on ICa-L, IK or IKur. CONCLUSION: These results support the idea that some of the electrical disturbances seen in hypothyroid hearts, such as the Ito and IK1 current reduction, could be caused not by low T3 but by the elevation of circulating TSH.

A simple methodology for characterization of germanium coaxial detectors by using Monte Carlo simulation and evolutionary algorithms.

The determination in a sample of the activity concentration of a specific radionuclide by gamma spectrometry needs to know the full energy peak efficiency (FEPE) for the energy of interest. The difficulties related to the experimental calibration make it advisable to have alternative methods for FEPE determination, such as the simulation of the transport of photons in the crystal by the Monte Carlo method, which requires an accurate knowledge of the characteristics and geometry of the detector. The characterization process is mainly carried out by Canberra Industries Inc. using proprietary techniques and methodologies developed by that company. It is a costly procedure (due to shipping and to the cost of the process itself) and for some research laboratories an alternative in situ procedure can be very useful. The main goal of this paper is to find an alternative to this costly characterization process, by establishing a method for optimizing the parameters of characterizing the detector, through a computational procedure which could be reproduced at a standard research lab. This method consists in the determination of the detector geometric parameters by using Monte Carlo simulation in parallel with an optimization process, based on evolutionary algorithms, starting from a set of reference FEPEs determined experimentally or computationally. The proposed method has proven to be effective and simple to implement. It provides a set of characterization parameters which it has been successfully validated for different source-detector geometries, and also for a wide range of environmental samples and certified materials.

Yessotoxin induces ER-stress followed by autophagic cell death in glioma cells mediated by mTOR and BNIP3.

Yessotoxin at nanomolar concentrations can induce programmed cell death in different model systems. Paraptosis-like cell death induced by YTX in BC3H1 cells, which are insensitive to several caspase inhibitors,has also been reported. This makes yessotoxin of interest in the search of molecules that target cancer cells vulnerabilities when resistance to apoptosis is observed. To better understand the effect of this molecule at the molecular level on tumor cells, we conducted a transcriptomic analysis using 3 human glioma cell lines with different sensitivities to yessotoxin. We show that the toxin induces a deregulation of the lipid metabolism in glioma cells as a consequence of induction of endoplasmic reticulum stress. The endoplasmic reticulum stress in turn arrests the cell cycle and inhibits the protein synthesis. In the three cell lines used we show that YTX induces autophagy, which is involved in cell death. The sensibility of the cell lines used towards autophagic cell death was related to their doubling time, being the cell line with the lowest proliferation rate the most resistant.The involvement of mTOR and BNIP3 in the autophagy induction was also determined.

Mechanism of cytotoxic action of crambescidin-816 on human liver-derived tumour cells.

BACKGROUND AND PURPOSE: Marine sponges have evolved the capacity to produce a series of very efficient chemicals to combat viruses, bacteria, and eukaryotic organisms. It has been demonstrated that several of these compounds have anti-neoplastic activity. The highly toxic sponge Crambe crambe has been the source of several molecules named crambescidins. Of these, crambescidin-816 has been shown to be cytotoxic for colon carcinoma cells. To further investigate the potential anti-carcinogenic effect of crambescidin-816, we analysed its effect on the transcription of HepG2 cells by microarray analysis followed by experiments guided by the results obtained. EXPERIMENTAL APPROACH: After cytotoxicity determination, a transcriptomic analysis was performed to test the effect of crambescidin-816 on the liver-derived tumour cell HepG2. Based on the results obtained, we analysed the effect of crambescidin-816 on cell-cell adhesion, cell-matrix adhesion, and cell migration by Western blot, confocal microscopy, flow cytometry and transmission electron microscopy. Cytotoxicity and cell migration were also studied in a variety of other cell lines derived from human tumours. KEY RESULTS: Crambescidin-816 had a cytotoxic effect on all the cell lines studied. It inhibited cell-cell adhesion, interfered with the formation of tight junctions, and cell-matrix adhesion, negatively affecting focal adhesions. It also altered the cytoskeleton dynamics. As a consequence of all these effects on cells crambescidin-816 inhibited cell migration. CONCLUSIONS AND IMPLICATIONS: The results indicate that crambescidin-816 is active against tumour cells and implicate a new mechanism for the anti-tumour effect of this compound.

ESX-1-induced apoptosis is involved in cell-to-cell spread of Mycobacterium tuberculosis.

Apoptosis modulation is a procedure amply utilized by intracellular pathogens to favour the outcome of the infection. Nevertheless, the role of apoptosis during infection with Mycobacterium tuberculosis, the causative agent of human tuberculosis, is subject of an intense debate and still remains unclear. In this work, we describe that apoptosis induction in host cells is clearly restricted to virulent M. tuberculosis strains, and is associated with the capacity of the mycobacteria to secrete the 6 kDa early secreted antigenic target ESAT-6 bothunder in vitro and in vivo conditions. Remarkably, only apoptosis-inducing strains are able to propagate infection into new cells, suggesting that apoptosis is used by M. tuberculosis as a colonization mechanism. Finally, we demonstrate that in vitro modulation of apoptosis affects mycobacterial cell-to-cell spread capacity, establishing an unambiguous relationship between apoptosis and propagation of M. tuberculosis. Our data further indicate that BCG and MTBVAC vaccines are inefficient in inducing apoptosis and colonizing new cells, correlating with the strong attenuation profile of these strains previously observed in vitro and in vivo.

Natural radioactivity measurements of beach sands in Gran Canaria, Canary Islands (Spain).

Concentrations of natural radionuclides (226)Ra, (232)Th, (40)K and man-made (137)Cs in most important tourist Gran Canaria beaches have been determined using a high-purity Germanium detector to analyse their radiological hazard. Average values of the activity concentrations of (226)Ra, (232)Th and (40)K were 17.6±1.4, 21.3±1.8 and 480±22 Bq kg(-1), respectively. Observed activity concentration values of (137)Cs were practically negligible from a radiological protection point of view. The results of this paper were compared with others published in the bibliography for beaches and coastal sediments of different countries. The mean external air absorbed dose rate was 43.9±2.8 nGyh(-1), which resulted in an outdoor annual effective dose below the world average. Also, the radium equivalent and the external hazard index were calculated. Results from Gran Canaria beaches showed the low levels of radioactivity, indicating no significant radiological risk related to human activities in the area.

Resveratrol inhibits proliferation of primary rat hepatocytes in G0/G1 by inhibiting DNA synthesis.

Resveratrol is a phytoalexin that has been shown to inhibit cell proliferation of several cancer cell lines. In some cases this inhibition was specific for the transformed cells when compared with normal cells of the same tissue. To test whether this was the case in rat hepatocytes, we exposed primary rat hepatocytes in culture and transformed rat hepatic cells to this compound and studied its effect on cell proliferation, measuring deoxy-bromouridine incorporation and total DNA. We also studied the effect of resveratrol on the cell cycle of normal and transformed rat hepatocytes. We observed that resveratrol inhibited proliferation in a dose-dependent manner in both cases, with no differential action in the transformed cells compared to the normal ones. This compound arrested the cell cycle in G0/G1 in primary hepatocytes, while it arrested the cell cycle in G2/M in transformed cells. Transformed hepatocytes showed accumulation of cells in the S phase of the cell cycle.

Characterization and activity determination of the human protein phosphatase 2A catalytic subunit α expressed in insect larvae.

Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit α of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit α of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250 µg per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94 µmol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2 months at -20 °C.

Comparative study of toxicological and cell cycle effects of okadaic acid and dinophysistoxin-2 in primary rat hepatocytes.

AIMS: To determine the relative toxicity and effects on the cell cycle of okadaic acid and dinophysistoxin-2 in primary hepatocyte cultures. MAIN METHODS: Cytotoxicity was determined by the MTT method, caspase-3 activity and lactate dehydrogenase release to the medium. The cell cycle analysis was performed by imaging flow cytometry and the effect of the toxins on cell proliferation was studied by quantitative PCR and confocal microscopy. KEY FINDINGS: We show that dinophysistoxin-2 is less toxic than okadaic acid for primary hepatocytes with a similar difference in potency as that observed in vivo in mice after intraperitoneal injection. Both toxins induced apoptosis with caspase-3 increase. They also inhibited the hepatocytes cell cycle in G1 affecting diploid cells and diploid bi-nucleated cells. In proliferating hepatocytes exposed to the toxins, a decrease of p53 gene expression as well as a lower protein level was detected. Studies of the tubulin cytoskeleton in toxin treated cells, showed nuclear localization of this molecule and a granulated tubulin pattern in the cytoplasm. SIGNIFICANCE: The results presented in this work show that the difference in toxicity between dinophysistoxin-2 and okadaic acid in cultured primary hepatocytes is the same as that observed in vivo after intraperitoneal injection. Okadaic acid and dinophysistoxin-2 arrest the cell cycle of hepatocytes at G1 even in diploid bi-nucleated cells. p53 and tubulin could be involved in the cell cycle inhibitory effect.