Constitutive expression of pea Lhcb 1–2 in tobacco affects plant development, morphology and photosynthetic capacity

Lhcb1–2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1–2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25°C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4°C for 4h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1–2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.     

Separation of cobalt binding proteins by immobilized metal affinity chromatography

BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 microl) was developed. Sample preparation involved a liquid-liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80-2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples.     

Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.     

Contrasting patterns of spatial genetic structure of diploid and triploid populations of the clonal aquatic species,<Emphasis Type="Italic">Butomus umbellatus (Butomaceae)</Emphasis>, in Central Europe

Genetic diversity in a sample of an aquatic plantButomus umbellatus from 37 localities in Czechia and Slovakia was studied by analyzing six polymorphic loci in three enzymatic systems (SKDH, PGD and AAT). Diversity among ramets was low in eight populations with relatively extensive sampling (only one population possessed more than one multilocus genotype), suggesting high clonality of reproduction in these populations. However, among-population diversity was high: G = 0.782 and 0.881 for the samples of diploid and triploid populations, respectively. Heterozygosity of individual plants averaged over variable loci was also high: H = 0.554 for diploids and 0.453 for triploids. Genetic differentiation among populations was additionally studied using cluster analysis. Several populations of diploids clustered separately from all other populations, whereas another group of diploid populations clustered with some triploid populations, indicating the possibility of relatively recent, probably multiple origin of these triploid populations from their diploid progenitors. Association between matrices of Nei’s genetic distances among populations from different localities and matrices of geographic distances among these localities revealed highly significant correlation for the sample of diploid populations (r = 0.60,P < 0.001) but no significant correlation for the sample of triploid populations (r = 0.02,P = 0.593). These results indicate a spatial structure of diploid populations in accordance with the isolation by distance model, and a random distribution of genotypes among triploid populations ofB. umbellatus.     

Effect of arsenic trioxide on metallothionein and its conversion to different arsenic metabolites in hen liver

The metabolism of arsenic, its affinity to metallothionein (MT), its influence on selenium levels, and its biotransformation to different metabolites in the liver tissue of laying hens exposed to arsenic trioxide (As2O3) was investigated. The experiment was performed with two groups of hens fed for 19 d with either a standard diet or with the same diet enriched in arsenic (30 microg/g). The major findings were as follows: 1. After 19 d exposure, about 65% of the total liver As was found in the water-soluble phase (100,000g centrifuged supernatant). In liver supernatant, As binding was found mostly in the range of very low-molecular-weight proteins (Mr < 10,000). Although after exposure the amount of MT-like proteins increased, the As bound to it was only in trace amounts. The protein was identified by convential procedures as Zn,Cu-thionein with traces of selenium and arsenic. 2. Arsenic exposure resulted in almost unchanged Se levels regarding its tissue concentrations and distribution between supernatant and pellet, where about 10% of total Se was found in the supernatant. On the contrary, As exposure did affect Cd levels. Tissue Cd concentration was slightly diminished, but the percentage of tissue Cd found in the water-soluble phase was increased from 20% to 40%. 3. In methanol extracts of tissue and supernatant of the As-exposed group, only two arsenic compounds were detected, As(III) and dimethylarsinic acid (DMA), the latter prevailing.     

Seasonal variation in seed predation by adult carabid beetles

In many agro‐ecosystems of the temperate region, granivorous ground beetles (Coleoptera: Carabidae) are the most important invertebrate predators of seeds after dispersal. The consumption of a particular species of seed differs among carabid species, but seasonal variation in consumption has not yet been studied. Seasonal differences in the quantity of seed consumed by the carabids Harpalus affinis (Schrank) and Harpalus distinguendus (Duftschmid) were studied in a cafeteria experiment. Adults were collected on five dates between April and November and provided with seeds of 28 species of dicotyledonous herbaceous plants. Proportions eaten of the seeds offered (preferences) were nearly identical in both carabid species. Overall seed consumption increased from early spring until late June, then gradually decreased towards autumn. Despite this variation, the preferences on particular dates were essentially similar. Seasonal variation in carabid physiology and feeding experience thus influenced overall seed consumption, but not their preferences for particular seed species.     

Native and alien floras in urban habitats: a comparison across 32 cities of central Europe

Aim To determine relative effects of habitat type, climate and spatial pattern on species richness and composition of native and alien plant assemblages in central European cities. Location Central Europe, Belgium and the Netherlands. Methods The diversity of native and alien flora was analysed in 32 cities. In each city, plant species were recorded in seven 1‐ha plots that represented seven urban habitat types with specific disturbance regimes. Plants were classified into native species, archaeophytes (introduced before ad 1500) and neophytes (introduced later). Two sets of explanatory variables were obtained for each city: climatic data and all‐scale spatial variables generated by analysis of principal coordinates of neighbour matrices. For each group of species, the effect of habitat type, climate and spatial variables on variation in species composition was determined by variation partitioning. Responses of individual plant species to climatic variables were tested using a set of binomial regression models. Effects of climatic variables on the proportion of alien species were determined by linear regression. Results In all cities, 562 native plant species, 188 archaeophytes and 386 neophytes were recorded. Proportions of alien species varied among urban habitats. The proportion of native species decreased with increasing range and mean annual temperature, and increased with increasing precipitation. In contrast, proportions of archaeophytes and neophytes increased with mean annual temperature. However, spatial pattern explained a larger proportion of variation in species composition of the urban flora than climate. Archaeophytes were more uniformly distributed across the studied cities than the native species and neophytes. Urban habitats rich in native species also tended to be rich in archaeophytes and neophytes. Main conclusions Species richness and composition of central European urban floras are significantly affected by urban habitat types, climate and spatial pattern. Native species, archaeophytes and neophytes differ in their response to these factors.     

Array comparative genome hybridization in patients with developmental delay: two example cases

Developmental delay is often a predictor of mental retardation (MR) or autism, two relatively frequent developmental disorders severely affecting intellectual and social functioning. The causes of these conditions remain unknown in most patients. They have a strong genetic component, but the specific genetic defects can only be identified in a fraction of patients. Recent developments in genomics supported the establishment of the causal link between copy number variants in the genomes of some patients and their affection. One of the techniques suitable for this analysis is array comparative genome hybridization, which can be used both for detailed mapping of chromosome rearrangements identified by classical cytogenetics and for the identification of novel submicroscopic gains or losses of genetic material. We illustrate the power of this approach in two patients. Patient 1 had a cytogenetically visible deletion of chromosome X and the molecular analysis was used to specify the gene content of the deletion and the prognosis of the child. Patient 2 had a seemingly normal karyotype and the analysis revealed a small recurrent deletion of chromosome 1 likely to be responsible for his phenotype. However, the genetic dissection of MR and autism is complicated by high heterogeneity of the genetic aberrations among patients and by broad variability of phenotypic effects of individual genetic defects.     

Prognostic Utility of Biomarkers in Predicting of One-Year Outcomes in Patients with Aortic Stenosis Treated with Transcatheter or Surgical Aortic Valve Implantation

Objectives

The aim of the work was to find biomarkers identifying patients at high risk of adverse clinical outcomes after TAVI and SAVR in addition to currently used predictive model (EuroSCORE).

Background

There is limited data about the role of biomarkers in predicting prognosis, especially when TAVI is available.

Methods

The multi-biomarker sub-study included 42 consecutive high-risk patients (average age 82.0 years; logistic EuroSCORE 21.0%) allocated to TAVI transfemoral and transapical using the Edwards-Sapien valve (n = 29), or SAVR with the Edwards Perimount bioprosthesis (n = 13). Standardized endpoints were prospectively followed during the 12-month follow-up.

Results

The clinical outcomes after both TAVI and SAVR were comparable. Malondialdehyde served as the best predictor of a combined endpoint at 1 year with AUC (ROC analysis) = 0.872 for TAVI group, resp. 0.765 (p<0.05) for both TAVI and SAVR groups. Increased levels of MDA, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase (TIMP1), ferritin-reducing ability of plasma, homocysteine, cysteine and 8-hydroxy-2-deoxyguanosine were all predictors of the occurrence of combined safety endpoints at 30 days (AUC 0.750–0.948; p<0.05 for all). The addition of MDA to a currently used clinical model (EuroSCORE) significantly improved prediction of a combined safety endpoint at 30 days and a combined endpoint (0–365 days) by the net reclassification improvement (NRI) and the integrated discrimination improvement (IDI) (p<0.05).Cystatin C, glutathione, cysteinylglycine, asymmetric dimethylarginine, nitrite/nitrate and MMP9 did not prove to be significant. Total of 14.3% died during 1-year follow-up.

Conclusion

We identified malondialdehyde, a marker of oxidative stress, as the most promising predictor of adverse outcomes during the 30-day and 1-year follow-up in high-risk patients with symptomatic, severe aortic stenosis treated with TAVI. The development of a clinical “TAVIscore” would be highly appreciated. Such dedicated scoring system would enable further testing of adjunctive value of various biomarkers.     

Global Changes in the Rat Heart Proteome Induced by Prolonged Morphine Treatment and Withdrawal

Morphine belongs among the most commonly used opioids in medical practice due to its strong analgesic effects. However, sustained administration of morphine leads to the development of tolerance and dependence and may cause long-lasting alterations in nervous tissue. Although proteomic approaches enabled to reveal changes in multiple gene expression in the brain as a consequence of morphine treatment, there is lack of information about the effect of this drug on heart tissue. Here we studied the effect of 10-day morphine exposure and subsequent drug withdrawal (3 or 6 days) on the rat heart proteome. Using the iTRAQ technique, we identified 541 proteins in the cytosol, 595 proteins in the plasma membrane-enriched fraction and 538 proteins in the mitochondria-enriched fraction derived from the left ventricles. Altogether, the expression levels of 237 proteins were altered by morphine treatment or withdrawal. The majority of changes (58 proteins) occurred in the cytosol after a 3-day abstinence period. Significant alterations were found in the expression of heat shock proteins (HSP27, α-B crystallin, HSP70, HSP10 and HSP60), whose levels were markedly up-regulated after morphine treatment or withdrawal. Besides that morphine exposure up-regulated MAPK p38 (isoform CRA_b) which is a well-known up-stream mediator of phosphorylation and activation of HSP27 and α-B crystallin. Whereas there were no alterations in the levels of proteins involved in oxidative stress, several changes were determined in the levels of pro- and anti-apoptotic proteins. These data provide a complex view on quantitative changes in the cardiac proteome induced by morphine treatment or withdrawal and demonstrate great sensitivity of this organ to morphine.