A landscape model for quantifying the trade-off between conservation needs and economic constraints in the management of a semi-natural grassland community

We present a landscape model to investigate the ecological consequences and costs of different management regimes in semi-natural grasslands. The model integrates dynamic abiotic conditions, management (i.e. disturbance) regime and response of more than 50 characteristic plant and insect species by modelling the dynamics of relevant niche parameters as predictors for species distribution models. We compare our results for exemplary scenarios differing in spatial and temporal scales and exemplary species belonging to different functional groups through several steps of aggregation.Our analysis aims at the question whether an infrequent massive disturbance by rototilling can serve as a less expensive alternative to annual mowing for preserving the characteristic species composition of open dry grasslands in Southern Germany. Rototilling results in a shifting mosaic determining the habitat quality for plant and animal species that may reduce the survival of local or regional populations.For some meadow species as well as the encroaching shrub species, rototilling has a detrimental effect on regional habitat quality. Other species, e.g. weeds and annual pioneers, strongly benefit or show only negligible reaction. Since this is a multi-objective problem, there is a no magic bullet in selecting an optimum scenario of measures. But by visualising the trade-off between ecological consequences and costs, our model is a valuable tool for conservation managers providing a sound scientific basis for management decisions relying on available ecological knowledge. It is also an interesting example for a model describing complex communities in a relatively simple way, simultaneously considering the main driving factors.     

Pomegranate phenolics from the peels, arils, and flowers are antiatherogenic: studies in vivo in atherosclerotic apolipoprotein e-deficient (E 0) mice and in vitro in cultured macrophages and lipoproteins

We have analyzed in vivo and in vitro the antiatherogenic properties and mechanisms of action of all pomegranate fruit parts: peels (POMxl, POMxp), arils (POMa), seeds (POMo), and flowers (POMf), in comparison to whole fruit juice (PJ). Atherosclerotic E 0 mice consumed POM extracts [200 microg of gallic acid equivalents (GAE)/mouse/day] for 3 months. Blood samples, peritoneal macrophages (MPM), and aortas were then collected. All POM extracts possess antioxidative properties in vitro. After consumption of PJ, POMxl, POMxp, POMa, or POMf by E (0) mice, the atherosclerotic lesion area was significantly decreased by 44, 38, 39, 6, or 70%, respectively, as compared to placebo-treated group, while POMo had no effect. POMf consumption reduced serum lipids, and glucose levels by 18-25%. PJ, POMxl, POMxp, POMf, or POMa consumption resulted in a significant decrement, by 53, 42, 35, 27, or 13%, respectively, in MPM total peroxides content, and increased cellular paraoxonase 2 (PON2) activity, as compared to placebo-treated mice. The uptake rates of oxidized-LDL by E (0)-MPM were significantly reduced by approximately 15% after consumption of PJ, POMxl, or POMxp. Similar results were obtained on using J774A.1 macrophage cell line. Finally, pomegranate phenolics (punicalagin, punicalin, gallic acid, and ellagic acid), as well as pomegranate unique complexed sugars, could mimic the antiatherogenic effects of pomegranate extracts. We conclude that attenuation of atherosclerosis development by some of the POM extracts and, in particular, POMf, could be related to the combined beneficial effects on serum lipids levels and on macrophage atherogenic properties.     

Latent and active polyphenol oxidase (PPO) in red clover (Trifolium pratense) and use of a low PPO mutant to study the role of PPO in proteolysis reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzyme's own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.     

Comparison of the key aroma compounds in organically grown, raw West-African peanuts (Arachis hypogaea) and in ground, pan-roasted meal produced thereof

Application of an aroma extract dilution analysis on an aroma distillate prepared from organically grown, raw West-African peanuts (Cameroon) revealed 36 odor-active areas in the flavor dilution (FD) factor range of 1 to 2048. The identification experiments, which were all performed by using the respective reference chemicals, revealed 2-isopropyl-3-methoxypyrazine (earthy, pea-like), 2-isobutyl-3-methoxypyrazine (bell pepper-like, earthy), and trans-4,5-epoxy-(E)-2-decenal (metallic) with the highest FD factors among the 36 aroma compounds identified. The two last mentioned odorants and another set of 22 further odorants were identified for the first time in raw peanuts. A comparative aroma extract dilution analysis applied on distillates prepared from either the raw peanuts or ground peanut meal roasted in a pan showed 52 odor-active areas in the FD factor range of 8 to 2048 in the roasted nut material. The identification experiments in combination with the FD factors revealed that among them, 2-acetyl-1-pyrroline and 4-hydroxy-2,5-dimethyl-3-(2H)-furanone showed the most significant contribution to the overall aroma, followed by 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, (E, E)-2,4-decadienal, and trans-4,5-epoxy-(E)-2-decenal. As a further result, 20 aroma compounds were newly identified in roasted peanuts, such as 2-propionyl-1-pyrroline and 2-acetyltetrahydropyridine (both popcorn-like). In particular, 2-acetyl-1-pyrroline and 4-hydroxy-2,5-dimethyl-3(2 H)-furanone showed the most pronounced increase after roasting.     

Use of isotope-labeled aflatoxins for LC-MS/MS stable isotope dilution analysis of foods

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.     

Simulated digestion and antioxidant activity of red wine fractions separated by high speed countercurrent chromatography

Wine is an important source of dietary antioxidants because of its phenolic compound content. The antioxidant activity (AA) of pure monomer substances present in wines, such as phenolic acids, flavanols, and anthocyanins, has already been described, but the AA of polymeric phenols is still unknown. In this study, we have fractionated a red wine by countercurrent chromatography (CCC) into four fractions: fraction 1, made up of polymeric compounds; fraction 2, containing malvidin-3-glucoside; fraction 3, containing peonidin-3-glucoside; and fraction 4, containing vitisin A. The AA of these fractions was determined by oxygen radical absorbance capacity and ferric reducing ability assays. The weight of fraction 1 was the largest, so this was the largest contributor to the AA of the wine. However, the antioxidant powers (muM Trolox/g fraction) of fractions 2-4 were similar and higher than that of fraction 1. We also determined AA before and after in vitro gastric and intestinal digestions. After gastric digestion, the AA was 100-1000 times higher than the original fraction values. Gallic acid was determined in gastric and intestinal digested fractions. After intestinal digestion, the concentrations of simple phenols, such as caffeic acid, p-coumaric acid, and protocatechualdehyde, increased as they were released from the fractions under our conditions. Protocatechuic acid was determined in more intestinal digested fractions than in gastric digested fractions. These results partly explain the increase in AA after the digestion and indicate the relevance of polymeric polyphenolic compounds as precursors of smaller molecules with biological activity.     

Diagnostic determination of melamine and related compounds in kidney tissue by liquid chromatography/tandem mass spectrometry

In 2007, it was determined that melamine, ammeline, ammelide, and cyanuric acid (abbreviated as MARC for melamine and related contaminants) had been added to wheat gluten and rice protein that were subsequently incorporated into pet food. The consumption of food tainted by MARC compounds was implicated in numerous instances of renal failure in cats and dogs. A method for the analysis of MARC compounds in kidney tissue using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) has been developed. MARC analytes were extracted by homogenization of kidney tissue in 50/40/10 acetonitrile/water/diethylamine. The homogenate was centrifuged, and an aliquot of supernatant was diluted with acetonitrile, concentrated, and fortified with a stable isotope-labeled analogue of melamine. Analytes were detected using atmospheric pressure chemical ionization and multiple reaction monitoring. Quantitation of positive samples was performed using the internal standard method and five-point calibration curves ranging between 50 and 1000 ng/mL of each analyte. The method was validated by analysis of replicate kidney tissue samples fortified with the individual analytes and by analysis of kidney samples fortified with melamine cyanurate powder at two different concentrations. This method was successfully used for routine postmortem diagnosis of melamine toxicosis in animals. Melamine was also detected by this method in paraffin-embedded tissue from animals suspected to have died of melamine toxicosis.     

Microorganisms as driving factors for the community structure of testate amoebae along an altitudinal transect in tropical mountain rain forests

We investigated microbial biomass, fungal biomass and microbial community structure at three altitudes (1000, 2000 and 3000 m) and in two soil layers [L/F layer (Layer I) and underlying H/Ah layer (Layer II)] of tropical mountain rain forests in southern Ecuador. Basal respiration, microbial biomass and concentration of ergosterol generally declined from Layer I to Layer II and peaked at 2000 m. Compared to temperate forest ecosystems microbial biomass and ergosterol concentrations were generally low. Patterns in phospholipid fatty acids indicated that the composition of microbial communities markedly changed from Layer I to Layer II. These differences between layers decreased with increasing altitude. The concentration of the arbuscular mycorrhizal fungal marker PLFA 16:1ω5c decreased with altitude in Layer I but increased in Layer II. The fungal-to-bacterial ratio increased with altitude and was higher in Layer I than in Layer II. Presumably, low microbial biomass in soils of tropical forest ecosystems is due to high temperature associated with high respiration but also low litter quality, with the latter declining with altitude. These conclusions are supported by the fact that at higher altitude the microbial community changed from a bacterial-dominated to a fungal-dominated system. CCA showed that microbial biomass correlated closely with density of a number of putatively bacterial feeding testate amoeba species including Corythion dubium Taranek, 1871, Euglypha cristata Leidy, 1879, Trigonopyxis arcula Penard, 1912, Tracheleuglypha dentata Deflandre, 1928 and Trinema lineare Penard, 1890. Ergosterol concentrations, but not the PLFA 18:2ω6c, strongly correlated with the putatively fungal feeding species Phryganella acropodia (Hertwig, Lesser, 1874) Hopkinson, 1909. Generally, parallel to microbial biomass and ergosterol concentrations the density of testate amoebae peaked at 2000 m. However, compared to microbial parameters changes in testate amoebae communities between two layers were less pronounced. The data suggest that density and community structure of testate amoebae are driven by the availability of food resources (bacteria and fungi) which at high altitude decrease with increasing moisture and decreasing pH.     

Optimization of an oxygen-based approach for community-level physiological profiling of soils

Current approaches for rapid assessment of carbon source utilization by whole soil communities (i.e., community-level physiological profiling or CLPP) provide a limited, biased view of microbial communities with little connection to in situ activities. We developed an alternative CLPP approach based upon fluorometric detection of dissolved oxygen consumption in a microtiter platform which offers flexible manipulation of experimental factors. In the attempt to reduce oxygen re-dissolution, the wells were filled with liquid to very near the top and sealed. We found that filling the wells with 240 vs. 150 μl of sample improved the sensitivity of the system to discern both the response to a substrate amendment as low as 10 mg l⁻¹ and un-amended, endogenous respiration. The preparation of a soil slurry facilitates inoculation into the microplate. Disruption of soil samples had a limited effect on the endogenous respiration in comparison to intact soil microbags in a 24-well microplate. Storage time (up to 33 days) reduced the level of activity in intact soil microbags but not in disrupted samples. A microcosm fertilization experiment was set to study the effects of N availability on the respiratory response in the plates. The use of soil organic carbon (SOC) and amended C-substrates (50 mg l⁻¹) was increased by the addition of nitrogen (N) in the plate, and appeared N-limited shortly after microcosm fertilization. The addition of the eukaryotic inhibitor cycloheximide delayed the initial increase in fluorescence (time to minimum response) of several C sources (casein, acetate, asparagine, coumaric acid), varying among soils, which could be explained by the fungal use of these compounds. However, the extent of the inhibition caused by cycloheximide did not increase at higher fungal to bacteria ratios as estimated by PLFA analysis, indicating that the direct estimation of the fungal biomass from cycloheximide addition is not feasible. This paper provides an optimized, standardized protocol for soil analysis, and sets the basis for further validation studies that will continue to define the underlying capabilities/biases of this approach.