Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.     

The physiological role of ascorbate as photosystem II electron donor: protection against photoinactivation in heat-stressed leaves

Previously, we showed that ascorbate (Asc), by donating electrons to photosystem II (PSII), supports a sustained electron transport activity in leaves in which the oxygen-evolving complexes were inactivated with a heat pulse (49°C, 40 s). Here, by using wild-type, Asc-overproducing, and -deficient Arabidopsis (Arabidopsis thaliana) mutants (miox4 and vtc2-3, respectively), we investigated the physiological role of Asc as PSII electron donor in heat-stressed leaves (40°C, 15 min), lacking active oxygen-evolving complexes. Chlorophyll-a fluorescence transients show that in leaves excited with trains of saturating single-turnover flashes spaced 200 ms apart, allowing continual electron donation from Asc to PSII, the reaction centers remained functional even after thousands of turnovers. Higher flash frequencies or continuous illumination (300 μmol photons m(-2) s(-1)) gradually inactivated them, a process that appeared to be initiated by a dramatic deceleration of the electron transfer from Tyr(Z) to P680(+), followed by the complete loss of charge separation activity. These processes occurred with half-times of 1.2 and 10 min, 2.8 and 23 min, and 4.1 and 51 min in vtc2-3, the wild type, and miox4, respectively, indicating that the rate of inactivation strongly depended on the Asc content of the leaves. The recovery of PSII activity, following the degradation of PSII proteins (D1, CP43, and PsbO), in moderate light (100 μmol photons m(-2) s(-1), comparable to growth light), was also retarded in the Asc-deficient mutant. These data show that high Asc content of leaves contributes significantly to the ability of plants to withstand heat-stress conditions.     

Factors driving spatial and temporal variation in production and production / biomass ratio of stream‐resident brown trout (Salmo trutta) in Cantabrian streams

1. The objective was to identify the factors driving spatial and temporal variation in annual production (P_A) and turnover (production/biomass) ratio (P/B_A) of resident brown trout Salmo trutta in tributaries of the Rio Esva (Cantabrian Mountains, Asturias, north‐western Spain). We examined annual production (total production of all age‐classes over a year) (P_A) and turnover (P/B_A) ratios, in relation to year‐class production (production over the entire life time of a year‐class) (P_T) and turnover (P/B_T) ratio, over 14 years at a total of 12 sites along the length of four contrasting tributaries. In addition, we explored whether the importance of recruitment and site depth for spatial and temporal variations in year‐class production (P_T), elucidated in previous studies, extends to annual production. 2. Large spatial (among sites) and temporal (among years) variation in annual production (range 1.9–40.3 g m^?2 per year) and P/B_A ratio (range 0.76–2.4 per year) typified these populations, values reported here including all the variation reported globally for salmonids streams inhabited by one or several species. 3. Despite substantial differences among streams and sites in all production attributes, when all data were pooled, annual (P_A) and year‐class production (P_T) and annual (P/B_A) and year‐class P/B_T ratios were tightly linked. Annual (P_A) and year‐class production (P_T) were similar but not identical, i.e. P_T = 0.94 P_A, whereas the P/B_T ratios were 4 + P/B_A ratios. 4. Recruitment (Rc) and mean annual density (N_A) were major density‐dependent drivers of production and their relationships were described by simple mathematical models. While year‐class production (P_T) was determined (R² = 70.1%) by recruitment (Rc), annual production (P_A) was determined (R² = 60.3%) by mean annual density (N_A). In turn, variation in recruitment explained R² = 55.2% of variation in year‐class P/B_T ratios, the latter attaining an asymptote at P/B_T = 6 at progressively higher levels of recruitment. Similarly, variations in mean annual density (N_A) explained R² = 52.1% of variation in annual P/B_A, the latter reaching an asymptote at P/B_A = 2.1. This explained why P/B_T is equal to P/B_A plus the number of year‐classes at high but not at low densities. 5. Site depth was a major determinant of spatial (among sites) variation in production attributes. All these attributes described two‐phase trajectories with site depth, reaching a maximum at sites of intermediate depth and declining at shallower and deeper sites. As a consequence, at sites where recruitment and mean annual density reached minimum or maximum values, annual (P_A) and year‐class production (P_T) and annual (P/B_A) and year‐class P/B_T ratios also reached minimum and maximum values.     

Alta prevalencia de obesidad en una población laboral en España

Background and objectivesTo report the prevalence of obesity in a Spanish working population and its changes in recent years.Material and methodsData were collected from routine medical examinations performed on workers by a national mutual insurance society for occupational accidents and diseases (Ibermutuamur). A structured questionnaire was completed and physical examinations were performed. Overweight was defined as BMI ranging from 25 and 29.9, obesity as BMI of 30-39.9, and morbid obesity as BMI ≥ 40 kg/m².ResultsData from 1,336,055 medical examinations performed from May 2004 to November 2007 were collected. Prevalence rates in the population examined in 2004 (n = 230,684; 73% males; average age, 36.4 years) were: morbid obesity, 0.5% (0.6% males, 0.5% females); obesity, 14.5% (17.0% males, 7.7% females); overweight, 38.4% (44.8% males, 21.3% females). Prevalence rates of obesity and overweight were higher in blue-collar workers (16.4% and 40.5% respectively) as compared to white-collar workers (10.9% and 34.4% respectively). There was a progressive increase in prevalence of obesity during the 4-year study (2004-2007) in both males (17.0%, 17.6%, 17.9%, 18.2%) and females (7.6%, 8.0%, 8.4%, 8.7%).ConclusionsPrevalence of obesity and overweight in the Spanish working population is high, especially in male blue-collar workers, and is increasing. There is a need to promote early prevention programs and specific treatments for obesity.     

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H⁺ with K⁺ and, to lesser extent, Na⁺. Here, we investigated the response to NaCl supply and K⁺ deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K⁺ the opposite was found. Analysis of mineral composition showed a higher K⁺ content in roots, shoots and xylem sap of transgenic plants and no differences in Na⁺ content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na⁺/H⁺ and, above all, K⁺/H⁺ transport activity in root intracellular membrane vesicles. Under K⁺ limiting conditions, transgenic plants enhanced root expression of the high‐affinity K⁺ uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K⁺ depletion rates and half cytosolic K⁺ activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K⁺ and lower cytosolic K⁺ activity than untransformed plants. These results indicate the fundamental role of K⁺ homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.     

PHOTOBIOTREATMENT: INFLUENCE OF NITROGEN AND PHOSPHORUS RATIO IN WASTEWATER ON GROWTH KINETICS OF SCENEDESMUS OBLIQUUS

Nitrogen and phosphorus concentration in the effluent of a wastewater treatment plant can vary significantly, which could affect the growth kinetic and chemical composition of microalgae when cultivated in this medium. The aim of this work was to study the rate of growth, nutrient removal and carbon dioxide biofixation as well as biomass composition of Scenedesmus obliquus (S. obliquus) when it is cultivated in wastewater at different nitrogen and phosphorus ratio, from 1:1 to 35:1. A more homogeneous method for calculating productivities in batch reactors was proposed. The proper N:P ratio for achieving optimum batch biomass productivity ranged between 9 and 13 (263 and 322 mg L^?1 d^?1 respectively). This was also the ratio range for achieving a total N and P removal. Above and below this range (9–13) the maximum biomass concentration changed, instead of the specific growth rate.The maximum carbon dioxide biofixation rate was achieved at N:P ratio between 13 and 22 (553 and 557 mg CO2 L^?1 d^?1 respectively). Lipid and crude protein content, both depend on the aging culture, reaching the maximum lipid content (34%) at the lowest N:P (1:1) and the maximum crude protein content (34.2%) at the highest N:P (35:1).     

Interactions Between Chronic Stress and Chronic Consumption of Caffeine on the Enzymatic Antioxidant System

We studied the effect of chronic caffeine on parameters related to oxidative stress in different brain regions of stressed and non-stressed rats. Wistar rats were divided into three groups: control (receiving water), caffeine 0.3 g/L and caffeine 1.0 g/L (in the drinking water). These groups were subdivided into non-stressed and stressed (repeated restraint stress during 40 days). Lipid peroxide levels and the total radical-trapping potential were assessed, as well as antioxidant enzyme activities superoxide dismutase, gluthatione peroxidase, and catalase in hippocampus, striatum and cerebral cortex. Results showed interactions between stress and caffeine, especially in the cerebral cortex, since caffeine increased the activity of some antioxidant enzymes, but not in stressed animals. We concluded that chronic administration of caffeine led, in some cases, to increased activity of antioxidant enzymes. However, these effects were not observed in the stressed animals.     

A tradeoff between protein stability and conformational mobility in homotrimeric dUTPases

Oligomerization directs active site formation in homotrimeric 2'-deoxyuridine triphosphate pyrophosphatases (dUTPases). Stability of the homotrimer is a central determinant in enzyme function. The present comparative studies of bacterial and fruitfly dUTPases with homologous 3D structures by differential scanning microcalorimetry; fluorescence, circular dichorism and infrared spectroscopies, demonstrate that unfolding is a two-state highly cooperative transition in both dUTPases excluding a significantly populated intermediate state of dissociated and folded monomers. The eukaryotic protein is much less resistant against either thermal or guanidine hydrochloride-induced denaturation. Results suggest that hydrophobic packing of the inner threefold channel of the dUTPase homotrimer greatly contributes to stability.     

Substrate specificity of DNA polymerases. I. Enzyme-catalysed incorporation of 5-''1-alkenyl)-2''-deoxyuridines into DNA.

A series of (E)-5-(1-alkenyl)-dUTPs as well as 5-vinyl-and (Z)-5-(1-propenyl)-dUTP have been synthesized to study steric requirements in DNA polymerase reactions. Experiments were carried out in E. coli DNA polymerase I Klenow fragment enzyme system. Substrates were characterized by KM and Vmax-values, initial incorporation rates as well as by total extent of incorporation of the analogues into poly(dA-dT) as a template-primer. Incorporation of the analogues could be best correlated with Vmax-values as well as the very similar initial incorporation rate values. Reactivity (Vmax/KM) showed no correlation with the extent of incorporation. 5-Vinyl-dUTP proved to be as good a substrate of the enzyme as dTTP, whereas (E)-5-(1-heptenyl)-and (E)-5-(1-octenyl)-dUTPs were very poor substrates, their incorporation was strongly limited and they also proved to be very efficient inhibitors of DNA replication, as shown by Ki-values. Substrate specificity of the Klenow enzyme can be explained by the steric hindrance of C-5 substituent, by the "orientational steric substituent effect" concept.     

Identification of Legionella spp. in Environmental Water Samples by ScanVIT-Legionella™ Method in Spain

Rapid and more sensitive methods for the detection and quantification of viable Legionella cells have been developed. In this paper, a comparative analysis of environmental water samples using the ScanVIT-Legionella? method and the traditional “gold standard” method of culturing is realised indicating the usefulness of the ScanVIT method. The ScanVIT-Legionella? method was performed on environmental water samples from different locations of Huesca region (Spain). Legionella micro-colonies should appear green colour and Legionella pneumophila micro-colonies appear red. Twenty-one environmental water samples analysed by standard culture plus five control samples (Two sterile water samples with Legionella as positive controls and three sterile water samples as negative controls). All of them were used to apply ScanVIT-Legionella? method. From of 21 environmental samples eleven were positive, six negative with both methods and four samples were negative for culture method and positive for ScanVIT-Legionella? method. The positive control samples were positive and the negative were negative for both methods. A comparative analysis of the results obtained with two methods showed a strong positive determination coefficient (R² = 0.99753). The results demonstrate the usefulness of the ScanVIT-Legionella? method as a rapid diagnostic tool in order to provide a diagnosis as quick as possible. ScanVIT-Legionella? method offers a series of advantages such as quickly diagnosis, higher sensitivity and the possibility to identify Legionella spp. and L. pneumophila simultaneously.