Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

Soil microbial diversity affects soil organic matter decomposition in a silty grassland soil

Soil microorganisms play a pivotal role in soil organic matter (SOM) turn-over and their diversity is discussed as a key to the function of soil ecosystems. However, the extent to which SOM dynamics may be linked to changes in soil microbial diversity remains largely unknown. We characterized SOM degradation along a microbial diversity gradient in a two month incubation experiment under controlled laboratory conditions. A microbial diversity gradient was created by diluting soil suspension of a silty grassland soil. Microcosms containing the same sterilized soil were re-inoculated with one of the created microbial diversities, and were amended with ¹³C labeled wheat in order to assess whether SOM decomposition is linked to soil microbial diversity or not. Structural composition of wheat was assessed by solid-state ¹³C nuclear magnetic resonance, sugar and lignin content was quantified and labeled wheat contribution was determined by ¹³C compound specific analyses. Results showed decreased wheat O-alkyl-C with increasing microbial diversity. Total non-cellulosic sugar-C derived from wheat was not significantly influenced by microbial diversity. Carbon from wheat sugars (arabinose-C and xylose-C), however, was highest when microbial diversity was low, indicating reduced wheat sugar decomposition at low microbial diversity. Xylose-C was significantly correlated with the Shannon diversity index of the bacterial community. Soil lignin-C decreased irrespective of microbial diversity. At low microbial diversity the oxidation state of vanillyl–lignin units was significantly reduced. We conclude that microbial diversity alters bulk chemical structure, the decomposition of plant litter sugars and influences the microbial oxidation of total vanillyl–lignins, thus changing SOM composition.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

How does drought stress influence the decomposition of plant litter with contrasting quality in a grassland ecosystem?

Background and aims

Plant litter quality and water availability both control decomposition. The interaction of both parameters was never studied. We used a grassland site, where litter of contrasting quality, i.e. green litter (fresh leaves; high quality) and brown litter (dead leaves, which underwent senescence but which are still attached to the plant; low quality), is returned to soil. Green and brown litter were exposed in the field under regular weather and drought conditions. The objective of this study was to evaluate the effect of drought on the decomposition of both litter types.

Methods

We incubated green and brown litter of three different grassland species (Lolium perenne, Festuca arundinacea and Dactylis glomerata) alone or as litter mixture (1/3 of each of the three grassland species) in litterbags for 28?weeks. Drought conditions were simulated by rainfall exclusion. After incubation, litter residues were analysed for C and nitrogen (N) content and stable isotope composition. Additionally, we determined the response of the lignin and carbohydrate signatures to the contrasting conditions.

Results

C decomposition kinetics of green and brown litter under drought conditions could be explained by two pools of contrasting turnover times. Drought decreased leaf litter C and N decomposition by more than 50% compared to regular weather conditions, mainly by strongly decreasing the decomposition rate constants. The lowest C decomposition occurred for mixtures of litter from all three grassland species. Brown litter showed on average 15% higher reduction in carbon decomposition than green litter following drought. Lignin content remained similar for green and brown litter after drought and regular weather conditions, while sugar content remained similar in green litter and decreased by 18% for brown litter under drought conditions.

Conclusions

Our results showed different response of decomposition of litter with contrasting quality to drought. Low quality brown litter is likely to be more affected than high quality green litter. Thus, litter quality must be taken into account, when assessing the effect of drought on decomposition.     

Assignment-free solution NMR method reveals CesT as an unswapped homodimer

The X-ray structure of the homodimeric chaperone CesT is the only structure among the type three secretion system (TTSS) chaperones that shows a domain swap. This swap has potential importance for the mechanism of effector translocation through a TTSS. Here we present two nuclear magnetic resonance strategies exploiting pre-existing structural models and residual dipolar couplings (RDCs), which reveal the unswapped 35.4-kDa dimer to be present in solution. Particularly efficient is the discrimination of a swapped and unswapped structural state performed simultaneously to automatic backbone assignment using only HN-RDCs and carbonyl backbone chemical shifts. This direct approach may prove to be generally useful to rapidly differentiate two structural models.     

Effects of drought and elevated temperature on biochemical composition of forage plants and their impact on carbon storage in grassland soil

Aims

The objective of this study was to investigate the effects of future warming and drought on (1) the biochemical composition of above-ground biomass of forage plants (Festuca arundinacea and Dactylis glomerata), (2) the potential mineralization of this material in soil, and (3) its priming effect on native soil organic matter.

Methods

We sampled above-ground plant material from spring regrowth and summer regrowth of a climate change experiment. While in spring, the plants were well watered, the summer regrowth was exposed to drought and elevated temperature (+3 °C) by infrared heating of the canopy during 3 weeks. We assessed the elemental and isotopic composition, lignin and non-cellulosic carbohydrate content and composition of plant material grown under all three conditions. Its mineralization potential in soil and priming effects were evaluated during laboratory incubation.

Results

Warming had no significant effect on elemental and stable isotope composition of both plant materials. In contrast, it resulted in reduction of lignin content for both plant species and decrease of the lignin-to-N ratio for F. arundinacea and increased non-cellulosic carbohydrate content for D. glomerata. Summer regrowth was characterised by increase of δ¹³C values, which is consistent with variations in stomatal conductance due to water shortage. Moreover, summer drought induced an increase in N content leading to decrease of the C/N ratio and increase of lignin-to-N ratio of summer regrowth compared to spring regrowth. Differences in decomposition were small, while priming effects were more strongly altered by the different exposure to enviromental.

Conclusion

Our results provide direct experimental evidence that extreme climatic events (high temperature and precipitation deficit) have an influence on soil carbon storage particularly through their effect on priming of native soil organic matter induced by altered plant litter. These effects seem to be governed by alterations of stoichiometry and to a smaller extent by alterations of plant chemical composition.     

Transformation of buffalo manure by composting or vermicomposting to rehabilitate degraded tropical soils

The addition of composted buffalo manure may lead to qualitative and quantitative improvement of the organic matter content of degraded tropical agricultural soils in Northern Vietnam. The objectives of this study were to follow the biochemical changes occurring during composting of buffalo manure with and without earthworms during 3 months and to study the effect of the end products (compost and vermicompost) on soil biochemical parameters and plant growth after two months of incubation under controlled conditions in an open pot experiment. Our conceptual approach included characterisation of organic matter of the two composts before and after addition to soil by elemental, isotopic analysis and analytical pyrolysis and comparison with conventional fertilisation. We also analysed for lignin content and composition.Our results showed that composting in the presence of earthworms led to stronger transformation of buffalo manure than regular composting. Vermicompost was enriched in N-containing compounds and depleted in polysaccharides. It further contained stronger modified lignin compared to regular compost. In the bulk soil, the amendment of compost and vermicompost led to significant modification of the soil organic matter after 2 months of exposure to natural weather conditions. The lignin component of SOM was unaffected whatever the origin of the organic amendment. Compost and vermicompost amendments both enhanced aggregation and increased the amount of organic matter in water stable aggregates. However, vermicompost is preferable to compost due to its beneficial effect on plant growth, while having similar positive effects on quantity and quality of SOM.