Effect of chilling on the diurnal rhythm of enzymes involved in protection against oxidative stress in a chilling-tolerant and a chilling-sensitive maize genotype

The effect of chilling on diurnal changes in activity of adenosine 5'-phosphosulfate sulfotransferase, glutathione reductase (EC 1.6.4.2) and glutathione transferase (EC 2.5.1.18) was analysed in the second leaf of Z 7, a chilling-tolerant, and Penjalinan, a chilling-sensitive maize (Zea mays L.) genotype. Nitrate reductase (EC 1.6.6.1) was measured for comparison. All enzyme activities examined changed with a typical diurnal rhythm in both genotypes cultivated at 25°C. Adenosine 5'-phosphosulfate sulfotransferase and nitrate reductase activity peaked during the light period, then decreased and reached lowest levels at the end of the dark period. Glutathione reductase activity increased in the dark and decreased during the light period. Maximum glutathione transferase activities were measured in the middle of the light period, minimal ones in the middle of the dark period. At 12°C these diurnal changes were eliminated in all enzymes examined of both genotypes.
The average adenosine 5'-phosphosulfate sulfotransferase and glutathione reductase activity were higher in the chilling-tolerant Z 7 than in the sensitive Penjanilan at 12°C in the light. Increased levels of both enzymes may contribute in establishing increased levels of cysteine and reduced glutathione in the chilling-tolerant Z 7. Indeed it has been shown before that the chilling-tolerant maize genotypes contain higher levels of both compounds at low temperatures than chilling-sensitive ones.     

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.     

Non‐symbiotic nitrogen fixation during leaf litter decomposition in an old‐growth temperate rain forest of Chiloé Island,southern Chile: Effects of single versus mixed species litter

Heterotrophic nitrogen fixation is a key ecosystem process in unpolluted, temperate old‐growth forests of southern South America as a source of new nitrogen to ecosystems. Decomposing leaf litter is an energy‐rich substrate that favours the occurrence of this energy demanding process. Following the niche ‘complementarity hypothesis’, we expected that decomposing leaf litter of a single tree species would support lower rates of non‐symbiotic N fixation than mixed species litter taken from the forest floor. To test this hypothesis we measured acetylene reduction activity in the decomposing monospecific litter of three evergreen tree species (litter C/N ratios, 50–79) in an old‐growth rain forest of Chiloé Island, southern Chile. Results showed a significant effect of species and month (anova , Tukey's test, P < 0.05) on decomposition and acetylene reduction rates (ARR), and a species effect on C/N ratios and initial % N of decomposing leaf litter. The lowest litter quality was that of Nothofagus nitida (C/N ratio = 78.7, lignin % = 59.27 ± 4.09), which resulted in higher rates of acetylene reduction activity (mean = 34.09 ± SE = 10.34 nmol h^?1 g^?1) and a higher decomposition rate (k = 0.47) than Podocarpus nubigena (C/N = 54.4, lignin % = 40.31 ± 6.86, Mean ARR = 4.11 ± 0.71 nmol h^?1 g^?1, k = 0.29), and Drimys winteri (C/N = 50.6, lignin % = 45.49 ± 6.28, ARR = 10.2 ± 4.01 nmol h^?1 g^?1, k = 0.29), and mixed species litter (C/N = 60.7, ARR = 8.89 ± 2.13 nmol h^?1g^?1). We interpret these results as follows: in N‐poor litter and high lignin content of leaves (e.g. N. nitida) free‐living N fixers would be at competitive advantage over non‐fixers, thereby becoming more active. Lower ARR in mixed litter can be a consequence of a lower litter C/N ratio compared with single species litter. We also found a strong coupling between in situ acetylene reduction and net N mineralization in surface soils, suggesting that as soon N is fixed by diazotroph bacteria it may be immediately incorporated into mineral soil by N mineralizers, thus reducing N immobilization.     

Sulfate Reduction Potential in Sediments in the Norilsk Mining Area,Northern Siberia

Abstract

The purpose of this study was to characterize the distribution and activity of sulfate-reducing bacteria in tailings and sediments impacted by effluents from mining and smelting operations in the Norilsk area in northern Siberia. The Norilsk mining complex involves three smelter operations, a hydrometallurgical plant, and extensive tailings areas located in the permafrost zone. Sulfate reduction rates measured with a ³⁵SO₄ ^2? tracer technique under various in-situ conditions ranged from 0.05 to 30 nmol S cm^?3 day^?1. Acetate and glucose addition greatly stimulated sulfate reduction, whereas lactate had less effect. The most pronounced stimulation of sulfate reduction (6.5-fold) was observed with phosphate amendment. Most-probable-number (MPN) counts of sulfate-reducing bacteria in media with glucose, ethanol, lactate, and acetate as electron donors were generally highest at around 10⁷ cells ml^?1. The actual MPN counts varied with the sample, electron donor, and incubation conditions (pH 7.2 vs. pH 3.5; 28°C vs. 4°C). Enrichment cultures of sulfate-reducing bacteria were established from a sample that showed the highest rate of sulfate reduction. After multiple serial transfers, the dominant sulfate-reducers were identified by fluorescence in situ hybridization using genus and group-specific 16S rRNA-targeted oligonucleotide probes. Desulfobulbus spp. prevailed in ethanol and lactate enrichments and the Desulfosarcina-Desulfococcus group dominated in acetate and benzoate enrichments. Psychrophilic Desulfotalea-Desulfofustis and moderately psychrophilic Desulforhopalus spp. were identified in enrichments incubated at 4°C, but they were also found in mesophilic enrichments.     

Nitrite reduction in Bacteroids of Rhizobium “hedysari” strain HCNT 1

Ex planta, bacteroids of the sulla-symbiont Rhizobium hedysari strain HCNT 1 terminated reduction of nitrite at nitrous oxide irrespective of the presence or absence of acetylene. Nitrate was not reduced during the experimental period, but slight nitrate reductase activity occurred if incubation with nitrate was prolonged (up to 15 h). As was observed in free-living cells, exposure of the bacteroids to the metal chelator, diethyldithiocarbamate, prevented reduction of nitrite, indicating the presence of a copper-containing nitrite reductase. Pulses of 10–75 M nitrite transiently impeded O₂ uptake in bacteroids, which resumed consumption of O₂ when the nitrite had been reduced. Exposure to >1.0 mM nitrite for 24h greatly inhibited nitrogenase activity (assayed as acetylene reduction activity) of bacteroids in planta. Exposure to the same concentrations of nitrite after 1h of incubation in the presence of acetylene almost completely stopped ongoing ethylene production in bacteroids of strain HCNT 1 extracted from nodules. Free cells of the non-nitrite-reducing R. hedysari strain CC 1335 were lacking in nitrogenase (acetylene-reduction) activity, whereas identically cultured (low-oxygen) strain HCNT 1 cells reduced both nitrite and acetylene.Abbreviations PMS phenazine methosulfate - DDC diethyldithiocarbamate     

Localization of iron-reducing activity in paddy soilby profile studies

Profiles of iron speciations (porewaterFe(II) and Fe(III), solid-phase Fe(II) andFe(III)) have been studied to localize both ironreduction and oxidation in flooded paddy soil. Sulfateand nitrate were determined to analyze interactions ofredox reactions involved in the iron cycle with thoseof the sulfur and nitrogen cycle. The development ofthe iron(II) and iron(III) profiles was observed inmicroscale over a time period of 11 weeks. After 11weeks the profiles were stable and showed lowestconcentrations of solid-phase iron(II) on the soilsurface with increasing concentrations to a soil depthof 10 mm ( 100 µmol/cm³). Profilesof iron(III) showed a maximum of iron(III) at a depthof 2 to 4 mm ( 100--200 µmol/cm³).Porewater iron(II) concentrations were three orders ofmagnitude lower than extracted iron(II) and indicatedthat most iron(II) was adsorbed to the solid-phase orimmobilized as siderite and vivianite. Diffusive lossof iron from the soil was indicated by iron recovery(0.3 µmol gdw^–1) in the flooding water after12 weeks. The organic content of the soil influencedthe concentrations of solid-phase iron(II) in deepersoil layers (> 6 mm); higher Fe(II) concentrationsin soil with limiting amounts of electron donors mayindicate lower consumption of CO₂ by methanogenicbacteria and therefore a higher sideriteprecipitation. Soil planted with rice showed similariron(II) profiles of fresh paddy soil cores. However,maximal iron(III) concentrations ( 350µmol/cm³) were present in planted soil at adepth of 1 to 2.5 mm where oxygen is provided by a matof fine roots. Sulfate and nitrate concentrations inthe porewater were highest on the soil surface (10µM NO₃ ^–, 40 µM SO₄ ^2–) anddecreased with depth. Similar profiles were detectedfor malate, acetate, lactate, and propionate, theconcentrations decreased gradually from the surface toa depth of 4 mm. Profiles of oxygen showed highestconcentrations at the surface due to photosyntheticproduction and a depletion of oxygen below 3 mm depth.Methane production rates measured from soil layersincubated separately in closed vessels were zero atthe soil surface and increased with depth. In soildepths below 4 mm where iron(III) concentrationsdecreased higher methane production rates werefound.     

Pelovirga terrestris gen. nov., sp. nov., anaerobic,alkaliphilic, fumarate-, arsenate-, Fe(III)- and sulfur-reducing bacterium isolated from a terrestrial mud volcano

A novel anaerobic, mesophilic, alkaliphilic, chemoorganotrophic bacterium (strain M08fum^T) was isolated from a salsa lake of a terrestrial mud volcano (Taman Peninsula, Russia). Cell of strain M08fum^T were Gram-stain-negative, rod-shaped, non-spore forming motile rods. The temperature range for growth was 10–45 °C (optimum at 30 °C). The pH range for growth was 7.0–11.0, with an optimum at pH 8.5–9.0. The isolate was capable of organic acids fermentation and anaerobic respiration with elemental sulfur, Fe(III) and arsenate. The end products of fumarate fermentation were succinate, acetate and CO₂. The closest phylogenetic relatives of strain M08fum^T were members of the family Geopsychrobacteraceae, class Desulfuromonadia. The genome of strain M08fum^T had a size of 3.10 Mb with a DNA G + C content of 53.1% (WGS). Genome analysis revealed the presence of genes involved in fumarate fermentation, arsenate reduction and resistance, sulfur respiration and Fe (III) reduction. Based on the phenotypic, genotypic and phylogenetic characteristics we propose to assign strain M08fum^T to a new species of a novel genus Pelovirga terrestris gen. nov., sp. nov. within the family Geopsychrobacteraceae. The type strain of Pelovirga terrestris is M08fum^T (=KCTC 15919^T = VKM B-3407^T). This is the first representative of the class Desulfuromonadia, isolated in pure culture from a mud volcano and the first alkaliphile in the family Geopsychrobacteraceae.     

Weak field influence on biomolecular changes

Model equations for the kinetics of the synthesis and decay of molecular aggregates are used to show the high sensitivity of equilibrium concentrations of high-molecular aggregates to external radiation. This phenomenon is used to explain the effects of low-intensity microwave fields on the functioning of biological systems. The experimental results on the influence of SHF-radiation on ferricyanide reduction by erythrocytes are interpreted in detail.     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

闭合复位与切开复位治疗桡骨远端骨折的疗效对比

目的:探讨闭合复位与切开复位对桡骨远端骨折患者的临床疗效。方法:回顾性分析2013年6月至2014年3月我院收治的60例桡骨远端骨折患者,随机数字表法分为切开复位组和闭合复位组,每组30例。闭合复位组患者给予闭合复位小夹板或石膏固定治疗,切开复位组患者给予切开复位内固定术治疗。观察并比较两组患者治疗前后掌倾角、尺偏角以及桡骨长度、术中出血量和手术时间,骨折愈合时间以及患者临床疗效进行检测并比较。结果:与治疗前相比,治疗后患者的掌倾角、尺偏角、桡骨长度水平均升高(P0.05);与闭合复位组相比,切开复位组患者掌倾角、尺偏角、桡骨长度评分水平较高(P0.05),术中出血量、手术时间水平较高(P0.05),临床治疗的优良率较高(P0.05),两组患者的骨折愈合时间无显著差异(P0.05)。结论:与闭合复位相比,切开复位能够明显恢复桡骨远端骨折患者的掌倾角、尺偏角以及桡骨长度,但手术时间以及术中出血量较多,临床疗效较好,两组患者的骨折愈合时间无明显差异。