Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen

Transgenic hybrid aspen (Populus tremula L. x P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.     

The Association of Cysteine with Obesity, Inflammatory Cytokines and Insulin Resistance in Hispanic Children and Adolescents

Context

Plasma total cysteine (tCys) independently relates to fat mass in adults. Dietary cyst(e)ine promotes adiposity and decreases glucose tolerance in some rodent models, but alleviates insulin resistance in others.

Objective

To investigate whether the association of tCys with body fat extends to children at particular risk of obesity, and whether tCys is associated with insulin resistance and obesity-associated inflammation.

Methods

We explored the cross-sectional relations of fasting plasma tCys and related metabolites with body composition measured by dual-energy X-ray absorptiometry in 984 Hispanic children and adolescents aged 4–19 years from the Viva La Familia Study. Linear and logistic regression and dose-response curves were used to evaluate relations of tCys with obesity, insulin resistance and inflammatory markers including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and C-reactive protein (CRP).

Results

tCys, methionine and total homocysteine (tHcy) increased with age. Upper tCys quartile was independently associated with a 5-fold increased risk of obesity (95% CI 3.5–8.0, P<0.001), and 2-fold risk of insulin resistance (95% CI: 1.6-5.0, P<0.001; adjusted for body fat%). Within the overweight/obese subgroup, but not in normal-weight children, tCys accounted for 9% of the variability in body fat% (partial r = 0.30, P<0.001; adjusted for age and gender). tCys correlated positively with serum non-esterified fatty acids and leptin, partly independent of body fat, but was not associated with serum IL-6, TNF-α or MCP-1. A positive correlation with CRP disappeared after adjustment for BMI.

Conclusion

tCys is independently associated with obesity and insulin resistance in Hispanic children and adolescents, highlighting a previously underappreciated link between the sulfur amino acid metabolic pathway and obesity and cardiometabolic risk.     

Communities of archaea and bacteria in a subsurface radioactive thermal spring in the Austrian Central Alps, and evidence of ammonia-oxidizing Crenarchaeota

Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (alpha, beta, gamma, and delta), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (delta-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.     

Nitrogen Cycling and Mass Balance for a Forested Catchment in the Italian Alps. Assessment of Nitrogen Status

During 1999–2001 the chemical composition and fluxes were measured in rainfall, throughfall, soil solution and stream water in a remote forested site in the Italian Alps. The analysis of temporal patterns revealed the differential behaviour of nitrogen and sulphur and suggested that different mechanisms controlled their flux. No important changes in sulphate concentration and fluxes emerged as the solution passed through the various components of the forest ecosystem, and temporal variations of SO₄ in the soil solution and stream were likely driven by the physical process of dilution. The availability of nitrate and ammonia, by contrast, was drastically reduced as throughfall water entered the soil and passed through the mineral layers, irrespective of season. The calculated hydrochemical budget based on throughfall and soil solution N fluxes revealed that ~80% N retention in the forest soil, corresponding to 12 kg ha⁻¹ yr⁻¹, despite a relatively high N deposition loading (15 kg ha⁻¹ yr⁻¹). Most of the leached nitrogen (90%) was in the organic form. Indicators of the N status of this ecosystem, such as C/N ratio in solid and solution phase of the soil and N foliage content as well as land use history were examined. Despite the strong N retention in the forested part of the catchment, the stream water N–NO₃ levels were consistently above 10 μg l⁻¹ suggesting that the Val Masino catchment as a whole was less efficient in processing atmospheric N inputs. This contrasting N behaviour illustrates the role of landscape features, such as the soil cover and vegetation type, that is characteristic of an alpine catchment.     

Time-dependent effects of sodium arsenite on DNA breakage and apoptosis observed in the comet assay

To assess genotoxic effects of sodium arsenite (NaAsO2) the single-cell gel electrophoresis (comet assay) had been conducted in various studies indicating genotoxicity. However, DNA fragmentation due to NaAsO2-induced apoptosis may constitute a bias in the interpretation of the results. Apoptotic cells can show typically large and diffuse comets, which are usually excluded during genotoxicity analysis. It is controversial whether there is a time-window in which the apoptotic process generates comets that would falsely be interpreted to be the result of genotoxic DNA damage. Therefore, we evaluated frequency histograms for single-cell measures of tail DNA (% DNA in comet tail) in 30-min intervals after incubation of mouse lymphoma L5178Y cells with sodium arsenite (NaAsO2). In parallel, we evaluated apoptosis by measuring annexin V-positive cells with flow cytometry, and visualized apoptotic cells on slides by Hoechst bisbenzimide 33258 staining. The first observed effect at 30 min after treatment was an increase in annexin V-positive cells. At about 60 min the number of cells with moderate DNA migration increased in the comet-assay analysis. After 90 min, an increase in the number of cells with high levels of DNA migration was observed, which resulted in a bimodal distribution of cells with moderate and high levels of DNA migration. Hoechst-stained apoptotic cells could only be observed at later times (> or = 120 min). This means that the treatment would have been considered to be genotoxic if analysed at 120 min even if the cells with high levels of DNA migration would have been excluded. The occurrence of annexin V-positive cells preceded the appearance of cells with moderate levels of DNA migration. We hypothesize that these cells were early apoptotic cells and not indicative of genotoxic damage. We conclude that DNA-damaging effects of NaAsO2 cannot adequately be interpreted if the comet assay is not accompanied by separate analysis of early endpoints for induction of apoptosis.     

Laboratory and field observations on the scuticociliate Histiobalantium from the pelagic zone of Lake Constance, FRG

Histiobalantium sp. was found regularly in the pelagic zoneof Lake Constance, FRG, over five annual cycles. Maxima of upto 6400 cells l^–1 were recorded in late summer, with similarnumbers in the 0–8 and 8–20 m depth intervals. Onan annual average, the population accounted for 10–17%of the total biomass of planktonic ciliates. In the laboratory,Histiobalantium grew well on a diet of the cryptophyte Rhodomonassp. Maximum growth rates obtained in batch cultures were 0.21and 0.33 day^–11 at 9 and 18°C, respectively. In situexperiments using diffusion chambers yielded positive growthrates in autumn and winter. The highest values recorded at theambient temperatures 5, 14 and 17°C were 0.17, 0.32 and0.40 day^–1, respectively. Comparing these results withthe different seasonal distributions and higher measured growthrates of other ciliates from Lake Constance, we conclude thatHistiobalantium is a superior competitor at relatively low algalfood concentrations. ²Present address: Fisheries & Oceans Canada, 4160 MarineDrive, West Vancouver, BC, V7V 1N6, Canada     

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.     

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.     

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.