How are nitrogen availability,fine‐root mass,and nitrogen uptake related empirically? Implications for models and theory

Understanding the effects of global change in terrestrial communities requires an understanding of how limiting resources interact with plant traits to affect productivity. Here, we focus on nitrogen and ask whether plant community nitrogen uptake rate is determined (a) by nitrogen availability alone or (b) by the product of nitrogen availability and fine‐root mass. Surprisingly, this is not empirically resolved. We performed controlled microcosm experiments and reanalyzed published pot experiments and field data to determine the relationship between community‐level nitrogen uptake rate, nitrogen availability, and fine‐root mass for 46 unique combinations of species, nitrogen levels, and growing conditions. We found that plant community nitrogen uptake rate was unaffected by fine‐root mass in 63% of cases and saturated with fine‐root mass in 29% of cases (92% in total). In contrast, plant community nitrogen uptake rate was clearly affected by nitrogen availability. The results support the idea that although plants may over‐proliferate fine roots for individual‐level competition, it comes without an increase in community‐level nitrogen uptake. The results have implications for the mechanisms included in coupled carbon‐nitrogen terrestrial biosphere models (CN‐TBMs) and are consistent with CN‐TBMs that operate above the individual scale and omit fine‐root mass in equations of nitrogen uptake rate but inconsistent with the majority of CN‐TBMs, which operate above the individual scale and include fine‐root mass in equations of nitrogen uptake rate. For the much smaller number of CN‐TBMs that explicitly model individual‐based belowground competition for nitrogen, the results suggest that the relative (not absolute) fine‐root mass of competing individuals should be included in the equations that determine individual‐level nitrogen uptake rates. By providing empirical data to support the assumptions used in CN‐TBMs, we put their global climate change predictions on firmer ground.     

2-D DIGE analyses of enriched secretory lysosomes reveal heterogeneous profiles of functionally relevant proteins in leukemic and activated human NK cells

As part of the innate immune system, natural killer (NK) cells detect and lyse tumor and virus-infected cells without prior antigen-dependent recognition and expansion. To this end, they utilize dual-function organelles that combine properties of conventional lysosomes and exocytotic vesicles. Upon stimulation, these secretory lysosomes (SLs) release their cytotoxic molecules into the immunological synapse. In addition, several molecules associated with secretory vesicles become exposed on the plasma membrane. Recent studies often took advantage of the few established NK cell lines, for instance to analyze the exocytotic machinery associated with NK cell vesicles. NK cell lines and primary NK cells differ, however, substantially in the expression of "typical" surface receptors and their requirements to induce target cell lysis. Here, we directly compared the lysosomal compartments of different NK cell populations. We enriched SLs of two leukemic cell lines (YTS and NKL) and IL-2-expanded NK cells by subcellular fractionation and characterized their proteome by 2-D difference gel electrophoresis and MS. Although the overall protein composition of the lysosomal preparations was very similar and more than 90% of the proteins were present at comparable levels, we define a cell line-specific setup of functionally relevant proteins involved in antigen presentation and cytotoxic effector function.     

The growth response of plants to elevated CO2 under non-optimal environmental conditions

Under benign environmental conditions, plant growth is generally stimulated by elevated atmospheric CO₂ concentrations. When environmental conditions become sub- or supra-optimal for growth, changes in the biomass enhancement ratio (BER; total plant biomass at elevated CO₂ divided by plant biomass at the current CO₂ level) may occur. We analysed literature sources that studied CO₂2environment interactions on the growth of herbaceous species and tree seedlings during the vegetative phase. For each experiment we calculated the difference in BER for plants that were grown under 'optimal' and 'non-optimal' conditions. Assuming that interactions would be most apparent if the environmental stress strongly diminished growth, we scaled the difference in the BER values by the growth reduction due to the stress factor. In our compilation we found a large variability in CO₂2environment interactions between experiments. To test the impact of experimental design, we simulated a range of analyses with a plant-to-plant variation in size common in experimental plant populations, in combination with a number of replicates generally used in CO₂2environment studies. A similar variation in results was found as in the compilation of real experiments, showing the strong impact of stochasticity. We therefore caution against strong inferences derived from single experiments and suggest rather a reliance on average interactions across a range of experiments. Averaged over the literature data available, low soil nutrient supply or sub-optimal temperatures were found to reduce the proportional growth stimulation of elevated CO₂. In contrast, BER increased when plants were grown at low water supply, albeit relatively modestly. Reduced irradiance or high salinity caused BER to increase in some cases and decrease in others, resulting in an average interaction with elevated CO₂ that was not significant. Under high ozone concentrations, the relative growth enhancement by elevated CO₂ was strongly increased, to the extent that high CO₂ even compensated in an absolute way for the harmful effect of ozone on growth. No systematic difference in response was found between herbaceous and woody species for any of the environmental variables considered.     

A MATHEMATICAL MODEL OF THROMBOPOIESIS IN RATS

A mathematical model of thrombopoiesis in rats is presented. This has four compartments; stem cells, megakaryocytes, thrombocytes and thrombopoietin. A high thrombopoietin concentration influences bone marrow proliferation in three ways. Firstly the stem cells are stimulated and a slow increase in megakaryocyte number follows. Secondly there are additional endomitoses in the (early) megakaryocytes resulting in an increase in megakaryocyte volume. Thirdly the megakaryocyte maturation time is shortened. The parameters of the model are determined from experimental values for the normal, maximum and minimum proliferation rates, maturation times and destruction rates. The model is tested by comparing simulated results for acute and chronic thrombocytopenia and thrombocytosis with experimental curves from the literature. The model and data agree within the limits of experimental error. Not all of the thrombopoietic regulatory system is known yet, so some important alternative hypotheses are investigated and compared with the model. Several hypotheses have been excluded in this way.     

ACTH-induced inhibition of endogenous rat brain protein phosphorylation in vitro: Structure activity

ACTH_1–24 inhibits the endogenous phosphorylation in vitro of distinct SPM protein bands. Using N-terminal fragments of ACTH, the structure-activity requirements for this effect were studied. A rather complex interaction of the ACTH fragments with endogenous SPM phosphorylation was observed. The effects were not only dependent on the primary structure of the peptide used, but also on the protein band studied and the ATP/SPM ratio used in the incubation system. ACTH_1–24 did not interfere with the ATP-hydrolyzing activity of the SPM preparation, nor did it influence the endogenous phosphatase activity. Therefore, a direct interaction of ACTH with SPM protein kinase(s) is likely to be responsible for its effect on phosphorylation.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

Habitat use of nightjar (Caprimulgus europaeus) in an Austrian pine forest

The Steinfeld in Lower Austria supports a population of European Nightjar (Caprimulgus europaeus) which was extensively studied during 1997 and 1998. The study area encompassed a pine forest of 20 km². The population densities of 1.05 and 1.25 territories/km², respectively, lies within the range found in central European populations. Annual monitoring until 2001 has shown the population to be stable. To gain an insight into habitat use of the species, various habitat-related parameters were measured inside and outside the territories, namely structure of trees, density of trees, structure of undergrowth vegetation and structure of clearings. Discriminant analysis was applied to assess the factors responsible for habitat choice of the Nightjar population. The findings showed that the Nightjars territories were frequently centered on a large clearing with an area of at least 0.7 ha. Clearings less than 50 m wide were not colonized. The requirement for a minimum width of a clearing in addition to a minimum area probably relates to better hunting conditions. Nightjars prefer trees where the lower edge of the crown is on average 4.38 m higher than at control points so that males can churr from dead branches immediately below the canopy. Such trees were found on the edge of clearings in the forest, and the edge of a clearing thus had a pronounced effect on the quality of a territory. In contrast to reports in the literature, neither the proportion of bare patches of ground nor the average height of undergrowth vegetation was found to be decisive for territory selection.     

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

Production of high amounts of 3-hydroxypropionaldehyde from glycerol by Lactobacillus reuteri with strongly increased biocatalyst lifetime and productivity

3-hydroxypropionaldehyde (3HPA) is a promising versatile substance derived from the renewable feedstock glycerol. It is a product of glycerol metabolism in Lactobacillus reuteri. Because of toxic effects, the biotechnological production is poor. In this work the biocatalyst lifetime and product formation could be drastically increased. In the established two-step process already applied, cells are grown in the first step under anaerobic conditions, and in the second step the immobilised or suspended biocatalyst is used for 3HPA-production under strict anaerobic conditions. In the first step it was possible to reach a biomass concentration of 5.5g CDW/L (OD(600)≈23.4). In the second step, normally, 3HPA accumulates to a toxic concentration and the reaction stops in less than 60min because of the interaction of 3HPA with cell components. To prevent this, the toxic product is bound to the newly found scavenger carbohydrazide to form the hydrazone. For the first time it was possible to recycle the immobilised biocatalyst for at least ten cycles (overall life time>33hours) in a repeated batch biotransformation with an overall production of 67g 3HPA. The optimal pH-value was between 6.8 and 7.2 at an optimal temperature of 40-45°C. In a single batch biotransformation with suspended resting cells it was possible to produce 150g/L 3HPA as carbohydrazone at an overall productivity of 10.7gL(-1)hours(-1). In a single fed-batch biotransformation at 45°C 138g/L glycerol was converted into 108g/L 3HPA with an overall productivity of 21.6gL(-1)hours(-1). This is the highest 3HPA concentration and productivities reported so far for the microbial production of 3HPA from glycerol.