Land use intensification in grasslands: higher trophic levels are more negatively affected than lower trophic levels

Increasing land use intensity and human influence are leading to a reduction in plant and animal species diversity. However, little is known about how these changes may affect higher trophic levels, apart from simply reducing species numbers. Here we investigated, over 3 years, the influence of different land practices on a tritrophic system in grassland habitats. The system consisted of the host plant Plantago lanceolata L. (Plantaginaceae), two monophagous weevils, Mecinus labilis Herbst and Mecinus pascuorum Gyllenhal (Coleoptera: Curculionidae), and their parasitoid Mesopolobus incultus Walker (Hymenoptera: Pteromalidae). At over 70 sites across three geographic regions in Germany, we measured plant species diversity and vegetation structure, as well as abundance of P. lanceolata, the two weevils, and the parasitoid. Land use intensity (fertilization) and type (mowing vs. grazing) negatively affected not only plant species richness but also the occurrence of the two specialized herbivores and their parasitoid. In contrast, land use had a mostly positive effect on host plant size, vegetation structure, and parasitization rate. This study reveals that intensification of land use influences higher trophic organisms even without affecting the availability of the host plant. The observed relationships between land use, vegetation complexity, and the tritrophic system are not restricted locally; rather they are measureable along a broad range of environmental conditions and years throughout Germany. Our findings may have important implications for the conservation of insect species of nutrient‐poor grasslands.     

Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A

Further development of our recently published Glu(pNA)-containing peptides (Anal. Biochem. 428 (2012) 73–80) provided new fluorogenic substrates for the activated blood coagulation factor XIII. A first series was designed by incorporation of Glu(AMC) at the penultimate position from the N terminus. For the best derivative H-Tyr-Glu(AMC)-Val-Lys-Val-Ile-NH₂, a moderate k_cat/K_m value of 34 s⁻¹ M⁻¹ was determined, which is more than 100-fold reduced compared with the previously reported Glu(pNA) substrates. Furthermore, two fluorescence resonance energy transfer (FRET) substrates were prepared by incorporation of an N-methyl-anthraniloyl fluorophore and a 2,4-dinitrophenyl quencher. Both substrates were excellently cleaved by FXIII-A₂^∗, which is generated from its zymogen by activation of thrombin in the presence of calcium ions. In the absence and presence of H-Gly-ethyl ester, k_cat/K_m values of 8010 and 8660 s⁻¹ M⁻¹, respectively, were found for the conversion of H-Lys(N(Me)Abz)-Glu(NH-(CH₂)₄-NH-Dnp)-Val-Lys-Val-Ile-Gly-NH₂ (substrate 8). These values are more than 200-fold improved compared with the Glu(AMC) substrates. Substrate 8 is suitable for the measurement of FXIII-A₂^∗ activities in plasma samples as well as for in vitro measurements. Furthermore, it was used for the determination of the inhibitory potency of a newly synthesized chloromethyl ketone derivative, Cbz-Phe-Glu(CMK)-Val-Lys-Val-Ile-Gly-NH₂, which was found to be a potent irreversible inhibitor of FXIII-A₂^∗.     

Visualization of cyclooxygenase-2 using a 2,3-diarylsubstituted indole-based inhibitor and confocal laser induced cryofluorescence microscopy at 20 K in melanoma cells in vitro

This study aimed at visualization of cyclooxygenase-2 (COX-2) protein expression in melanoma cells by confocal laser induced cryofluorescence microscopy using 4-(3-(4-methoxyphenyl)-1H-indol-2-yl)benzene-sulfonamide (C1) representative for a novel class of autofluorescent 2,3-diarylsubstituted indole-based selective COX-2 inhibitors.COX-2 expression was measured in human melanoma cell lines A2058 and MelJuso by immunocytochemistry and immunoblotting. Cellular uptake experiments using varying C1 concentrations down to 0.1 nM (with/without molar excess of celecoxib as control) were performed at 37 °C. Cryofluorescence microscopy was conducted at 20 K.COX-2 protein expression was successfully visualized by C1 in A2058 cells. COX-2-negative MelJuso cells showed no specific accumulation of C1. Control experiments using celecoxib and, additionally, implemented fluorescence spectroscopy confirmed specificity of both cellular uptake and intracellular association of C1.Cryofluorescence microscopy in combination with spectroscopy allowed for visualization of COX-2 protein expression in melanoma cells in vitro using a selective COX-2 inhibitor at very low concentrations.     

Cytoplasmic localization of PML particles in laminopathies

There is growing evidence that laminopathies, diseases associated with mutations in the LMNA gene, are caused by a combination of mechanical and gene regulatory distortions. Strikingly, there is a large variability in disease symptoms between individual patients carrying an identical LMNA mutation. This is why classical genetic screens for mutations appear to have limited predictive value for disease development. Recently, the widespread occurrence of repetitive nuclear ruptures has been described in fibroblast cultures from various laminopathy patients. Since this phenomenon was strongly correlated with disease severity, the identification of biomarkers that report on these rupture events could have diagnostic relevance. One such candidate marker is the PML nuclear body, a structure that is normally confined to the nuclear interior, but leaks out of the nucleus upon nuclear rupture. Here, we show that a variety of laminopathies shows the presence of these cytoplasmic PML particles (PML CPs), and that the amount of these protein aggregates increases with severity of the disease. In addition, between clinically healthy individuals, carrying LMNA mutations, significant differences can be found. Therefore, we postulate that detection of PML CPs in patient fibroblasts could become a valuable marker for diagnosis of disease development.