Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy

Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking, fluorescence resonance energy transfer imaging, sub-diffraction resolution microscopy and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm.     

Uptake of pulse injected nitrogen by soil microbes and mycorrhizal and non-mycorrhizal plants in a species-diverse subarctic heath ecosystem

HiMag tall fescue (Lolium arundinaceum (Schreb.) S.J. Darbyshire = Festuca arundinacea Schreb) was selected for high Mg concentration in the herbage to reduce grass tetany risk to ruminants; however, the mechanism of increased Mg uptake into shoots is unknown. The objective was to determine cation concentrations of roots, crowns, and leaves in plants of cv. HiMag and its parents, cv. Kentucky 31 and cv. Missouri 96, grown in nutrient solution for 42 days, and determine if cation ratios in roots, crowns, and leaves are different, indicating a difference due to translocation. Treatments were “basal” (1.5 mM K and 0.5 mM Mg), “K” (3.2 mM K), “Mg” (1 mM Mg), and “K?+?Mg” (3.2 mM K and 1 mM Mg). For HiMag, Mg was lower in roots (Trial 2 only), not different in crowns, and greater in leaves than Kentucky 31 and Missouri 96. Doubling the K and Mg of the nutrient solution from basal levels resulted in a 44% reduction of root Mg in Kentucky 31 and Missouri 96, compared to a 17% reduction in root Mg for HiMag. The K inflow rate in HiMag for the basal treatment was lower than that in Kentucky 31 and Missouri 96. These results provide evidence for a process that limits K uptake and an active Mg translocation mechanism in tall fescue. HiMag was apparently selected for traits that promote translocation of Mg from roots to shoots.     

Selective acylation enhances membrane charge sensitivity of the antimicrobial peptide mastoparan-x

The partitioning of the wasp venom peptide mastoparan-X (MPX) into neutral and negatively charged lipid membranes has been compared with two new synthetic analogs of MPX where the N^α-terminal of MPX was acylated with propanoic acid (PA) and octanoic acid (OA). The acylation caused a considerable change in the membrane partitioning properties of MPX and it was found that the shorter acylation with PA gave improved affinity and selectivity toward negatively charged membranes, whereas OA decreased the selectivity. Based on these findings, we hypothesize that minor differences in the embedding and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptide's selectivity between neutral and negatively charged lipid membranes. This finding is unique compared to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge—a finding that should be investigated for other, more potent antimicrobial peptides in future studies.     

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P(2)] is a newly identified phosphoinositide that modulates intracellular Ca(2+) by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca(2+), we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca(2+) stores PI(3,5)P(2) would increase intracellular Ca(2+) in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P(2) was present in the mouse aorta and that exogenously applied PI(3,5)P(2) readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P(2) elevated intracellular Ca(2+), and it also contracted aortic rings. Both the rise in intracellular Ca(2+) and the contraction caused by PI(3,5)P(2) were prevented by antagonizing RyRs, while the majority of the PI(3,5)P(2) response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca(2+) stores with thapsigargin or caffeine and/or ryanodine blunted the Ca(2+) response and greatly attenuated the contraction elicited by PI(3,5)P(2). The removal of extracellular Ca(2+) or addition of verapamil to inhibit voltage-dependent Ca(2+) channels reduced but did not eliminate the Ca(2+) or contractile responses to PI(3,5)P(2). We also found that PI(3,5)P(2) depolarized aortic smooth muscle cells and that LaCl(3) inhibited those aspects of the PI(3,5)P(2) response attributable to extracellular Ca(2+). Thus, full and sustained aortic contractions to PI(3,5)P(2) required the release of SR Ca(2+), probably via the activation of RyR, and also extracellular Ca(2+) entry via voltage-dependent Ca(2+) channels.     

Absence of Howlers (<Emphasis Type="Italic">Alouatta palliata</Emphasis>) Influences Tree Seedling Densities in Tropical Rain Forest Fragments in Southern Mexico

The disappearance of frugivorous primates in fragmented forests can potentially lower the rates of seed dispersal and recruitment of endozoochorous tree species, thus altering plant community structure. We quantified seedling density for 7 tree species that are common in the feces of mantled howlers (Alouatta palliata) in 6 rain forest fragments in northern Chiapas, Mexico. Howlers were present in 3 of the fragments and absent in the other 3. We compared seedling density in primate sleeping sites in inhabited fragments with control sites, which were structurally similar to sleeping sites but where we did not find monkey feces, in both inhabited and uninhabited fragments. For each tree species, we determined the relationship between seedling density and the local density of seeds and adult trees. In fragments where howlers were present, seedling density for 4 of the focal tree species (Brosimum alicastrum, Dialium guianense, Manilkara zapota, and Nectandra ambigens) was greater in sleeping sites than in control sites found in the same fragments. Moreover, seedling density of Dialium guianense was greater in the control sites of fragments inhabited by howlers than in fragments where this primate is absent. Seedling density of these 4 species correlates positively with seed density on the forest floor; however, we observed no correlations between seedling density and the density of adult trees. Our results suggest that the diversity of the seedling community of tree species dispersed by howlers may decline in fragments where this seed disperser is absent. These findings, together with the fact that only 5% of the study region is currently covered by forest and 81% of the forest remnants are uninhabited by mantled howlers, suggest that the potential long-term recovery of associated populations of tropical tree species dispersed by this primate species is highly uncertain. Conservation and restoration efforts should be aimed at restoring or replacing the ecological role played by this important seed disperser in the region.     

Antioxidant activity of synthetic cytokinin analogues: 6-alkynyl- and 6-alkenylpurines as novel 15-Lipoxygenase inhibitors

Synthetic cytokinin analogues as well as the well known CKs 6-benzylaminopurine (BAP), kinetin and trans-zeatin were examined for antioxidant activity. The compounds were tested as potential diphenylpicrylhydrazyl (DPPH) scavengers and as inhibitors of 15-lipoxygenase (15-LO). The natural plant hormones were essentially inactive in both assays, but several synthetic analogues have a profound inhibiting effect on 15-lipoxygenase from soybeans. The same compounds were only weak DPPH scavengers and they may therefore be regarded as so-called non antioxidant inhibitors of 15-LO.     

Estimation of Density-Dependent Mortality of Juvenile Bivalves in the Wadden Sea

We investigated density-dependent mortality within the early months of life of the bivalves Macoma balthica (Baltic tellin) and Cerastoderma edule (common cockle) in the Wadden Sea. Mortality is thought to be density-dependent in juvenile bivalves, because there is no proportional relationship between the size of the reproductive adult stocks and the numbers of recruits for both species. It is not known however, when exactly density dependence in the pre-recruitment phase occurs and how prevalent it is. The magnitude of recruitment determines year class strength in bivalves. Thus, understanding pre-recruit mortality will improve the understanding of population dynamics. We analyzed count data from three years of temporal sampling during the first months after bivalve settlement at ten transects in the Sylt-Rømø-Bay in the northern German Wadden Sea. Analyses of density dependence are sensitive to bias through measurement error. Measurement error was estimated by bootstrapping, and residual deviances were adjusted by adding process error. With simulations the effect of these two types of error on the estimate of the density-dependent mortality coefficient was investigated. In three out of eight time intervals density dependence was detected for M. balthica, and in zero out of six time intervals for C. edule. Biological or environmental stochastic processes dominated over density dependence at the investigated scale.     

N-Glycosylation of Asparagine 8 Regulates Surface Expression of Major Histocompatibility Complex Class I Chain-related Protein A (MICA) Alleles Dependent on Threonine 24

NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn⁸) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr²⁴) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.