Novel Approaches to Study Climate Change Effects on Terrestrial Ecosystems in the Field: Drought and Passive Nighttime Warming

This article describes new approaches for manipulation of temperature and water input in the field. Nighttime warming was created by reflection of infrared radiation. Automatically operated reflective curtains covered the vegetation at night to reduce heat loss to the atmosphere. This approach mimicked the way climate change, caused by increased cloudiness and increased greenhouse gas emissions, alters the heat balance of ecosystems. Drought conditions were created by automatically covering the vegetation with transparent curtains during rain events over a 2–5-month period. The experimental approach has been evaluated at four European sites across a climate gradient. All sites were dominated (more than 50%) by shrubs of the ericaceous family. Within each site, replicated 4-m × 5-m plots were established for control, warming, and drought treatments and the effect on climate variables recorded. Results over a two-year period indicate that the warming treatment was successful in achieving an increase of the minimum temperatures by 0.4–1.2°C in the air and soil. The drought treatment resulted in a soil moisture reduction of 33%–82% at the peak of the drought. The data presented demonstrate that the approach minimizes unintended artifacts with respect to water balance, moisture conditions, and light, while causing a small but significant reduction in wind speed by the curtains. Temperature measurements demonstrated that the edge effects associated with the treatments were small. Our method provides a valuable tool for investigating the effects of climate change in remote locations with minimal artifacts.     

A subnanosecond resolving spectrofluorimeter for the analysis of protein fluorescence kinetics

A spectrofluorometer is described consisting of an excitation source, optics, detector and time resolving electronics. The excitation source consists of a mode-locked Ar ion laser, synchronously pumps a dye laser, followed by a frequency doubling device. The repetition frequency of the U.V. pulses (FWHM some ps) has been reduced by an extra-cavity electro-optical modulator. Provisions have been made in the optical configuration to determine both time-resolved fluorescence spectra and fluorescence anisotropy decay curves. The commercially avialable electronics have been optimized for maximum time resolution. The spectral output of the excitation source is confined between 280 and 310 nm, which encompasses the region for eliciting protein fluorescence. The performance of the complete system has been tested with single lifetime standards line p-terphenyl in cyclohexane or with $\text{N-}\text{acetyl}\text{-}\text{L}\text{-}\text{tryptophanamide}$ in pH 7.5 buffer. Serum albumins from human and bovine sources have been employed as examples for time resolved fluorescence spectra and for the demonstration of anisotropy decay curves. Using these methods protein dynamics in the (sub)nanosecond time region can be directly explored.     

Genistein directly inhibits GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

The isoflavone-derivative genistein is commonly applied as an inhibitor of tyrosine kinases. In this report we analyze the effect of genistein on insulin-stimulated glucose uptake in 3T3-L1 adipocytes. In these cells insulin-induced glucose uptake is primarily mediated by the GLUT4 glucose transporter. We observed that pre-treatment with genistein did not affect insulin-induced tyrosine kinase activity of the insulin receptor or activation of protein kinase B. On the other hand, genistein acted as a direct inhibitor of insulin-induced glucose uptake in 3T3-L1 adipocytes with an IC(50) of 20 microM. We conclude that apart from acting as a general tyrosine kinase inhibitor, genistein also affects the function of other proteins such as the GLUT4 transporter. These data suggest that caution must be applied when interpreting data on the involvement of tyrosine kinase activity in glucose uptake in 3T3-L1 adipocytes.     

Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase

The highly conserved family of 14-3-3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 suggest functional isoform specificity of 14-3-3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14-3-3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14-3-3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14-3-3 isoforms. We found that 14-3-3C is unavailable in dark harvested barley leaf extract and 14-3-3A is functionally not capable to efficiently inhibit NR activity, leaving 14-3-3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14-3-3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14-3-3A to the alternative residue, as found in 14-3-3B and 14-3-3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14-3-3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14-3-3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14-3-3 isoforms as well as binding affinity determine isoform-specific regulation of NR activity.     

Applications of ecological stoichiometry for sustainable acquisition of ecosystem services

Human activities have differentially altered biogeochemical cycling at local, regional and global scales. We propose that a stoichiometric approach, examining the fluxes of multiple elements and the ratio between them, may be a useful tool for better understanding human effects on ecosystem processes and services. The different scale of impacts of the elements carbon, nitrogen and phosphorus and the different nature of their biogeochemical cycles, imply a large variation of their stoichiometric ratios in space and time and thus divergent impacts on biota.
In this paper, we examine the effects of anthropogenic perturbations on nutrient ratios in ecosystems in two examples and one case study. Altered stoichiometry in agricultural systems (example 1) can affect not only crop yield and quality but also the interactions between plants and their pollinators, pests and pathogens. Human activities have also altered stoichiometry in coastal ecosystems (example 2). Increased N loading has especially lead to increased N:P and reduced Si:N ratios, with detrimental effects on ecosystem services derived from coastal pelagic food webs, such as fish yield and water quality. The terrestrial–aquatic linkage in stoichiometric alterations is illustrated with a case study, the Mississippi River watershed, where anthropogenic activities have caused stoichiometric changes that have propagated through the watershed into the northern Gulf of Mexico.
Coupled with altered stoichiometric nutrient inputs are the inherent differences in variation and sensitivity of different ecosystems to anthropogenic disturbance. Furthermore, the connections among the components of a watershed may result in downstream cascades of disrupted functioning. Applying a multiple element perspective to understanding and addressing societal needs is a new direction for both ecological stoichiometry and sustainability.     

Effect of variable nutrient supply rates on the RNA level of a heterotrophic bacterial strain

The heterotrophic bacterial strain HIS 53 was grown in a continuous culture under chemostat conditions and at sinusoidal or stepwise variations of the dilution rate; aspartate, ammonium, and phosphate were the growth-limiting nutrients. Within a specific nutrient limitation the growth yield was constant and independent of the applied environmental conditions. Compared with the reference chemostat culture, sinusoidal variations of the dilution rate increased the cellular RNA level by 19%–53% dependent on the growth limitation; stepwise variations caused an increase of the RNA level by 28%–41%. It was hypothesized that under the variable environmental conditions in the natural habitat the physiological potential of the organism is enhanced by some such increase of the cellular RNA level. As a consequence these increased RNA levels influence the competition between heterotrophic bacteria and, as a result, also the composition of the population of heterotrophic bacteria.     

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.     

Optimality principles for model-based prediction of human gait

Although humans have a large repertoire of potential movements, gait patterns tend to be stereotypical and appear to be selected according to optimality principles such as minimal energy. When applied to dynamic musculoskeletal models such optimality principles might be used to predict how a patient's gait adapts to mechanical interventions such as prosthetic devices or surgery. In this paper we study the effects of different performance criteria on predicted gait patterns using a 2D musculoskeletal model. The associated optimal control problem for a family of different cost functions was solved utilizing the direct collocation method. It was found that fatigue-like cost functions produced realistic gait, with stance phase knee flexion, as opposed to energy-related cost functions which avoided knee flexion during the stance phase. We conclude that fatigue minimization may be one of the primary optimality principles governing human gait.