Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

A short LysM protein with high molecular diversity from an arbuscular mycorrhizal fungus,Rhizophagus irregularis

Arbuscular mycorrhizal (AM) fungi form an intimate symbiosis with roots of more than 80% of land plants without eliciting a significant defense response, and how they do so is yet to be determined. Typically, plants mount a defense response upon sensing chitin in fungal walls, and to counteract this response, plant-pathogenic fungi secrete small effector proteins with chitin-binding LysM domains. In the AM fungus, Rhizophagus irregularis, a small, putatively-secreted LysM protein, which we refer to as RiSLM, is among the most highly expressed effector-like proteins during symbiosis. Here, we show that RiSLM expression is reduced during non-functional symbiosis with Medicago mutants, mtpt4-2 and vapyrin. We demonstrate that RiSLM can bind to both chitin and chitosan, and we model the protein-ligand interaction to identify possible binding sites. Finally, we have identified RiSLM homologs in five published R. irregularis isolate genomes and demonstrate that the gene is subject to a high rate of evolution and is experiencing positive selection, while still conserving putative function. Our results present important clues for elucidating a role for a LysM effector, RiSLM, in AM symbiosis.     

Summer rainfall variability in European Mediterranean mountains from the sixteenth to the twentieth century reconstructed from tree rings

Since the end of the last glacial period, European Mediterranean mountains have provided shelter for numerous species of Eurosiberian and Boreal origin. Many of these species, surviving at the southern limit of their range in Europe and surrounded by Mediterranean ones, are relatively intolerant to summer drought and are in grave danger of loss, as a result of increasingly long and frequent droughts in this region. This is the case of the Scots pine (Pinus sylvestris) and the Austrian pine (Pinus nigra ssp. salzmannii) which are found on Central Iberian Peninsula at the edge of their natural range. We used a tree ring network of these two species to reconstruct past variations in summer rainfall. The reconstruction, based upon a tree ring composite chronology of the species, dates back to 1570 (adjusted R ²?=?0.49, P?<?0.000001) and captures interannual to decadal scale variability in summer precipitation. We studied the spatial representativeness of the rainfall patterns and described the occurrence rate of extremes of this precipitation. To identify associations between macroclimatic factors and tree radial growth, we employed a principal component analysis to calculate the resultant of the relationship between the growth data of both species, using this resultant as a dependent variable of a multiple regression whose independent variables are monthly mean temperature and precipitation from the average records. Spatial correlation patterns between instrumental precipitation datasets for southern Europe and reconstructed values for the 1950–1992 period indicate that the reconstruction captures the regional signal of drought variability in the study region (the origin of this precipitation is convective: thermal low pressure zones induced in the inland northeastern areas of the Iberian Peninsula). There is a clear increase in the recurrence of extreme dry events as from the beginning of twentieth century and an abrupt change to drier conditions. There appears to be a tendency toward recurrent exceptionally dry summers, which could involve a significant change for the Eurosiberian refugee species.     

No Evidence of Persisting Unrepaired Nuclear DNA Single Strand Breaks in Distinct Types of Cells in the Brain,Kidney, and Liver of Adult Mice after Continuous Eight-Week 50 Hz Magnetic Field Exposure with Flux Density of 0.1 mT or 1.0 mT

Background

It has been hypothesized in the literature that exposure to extremely low frequency electromagnetic fields (50 or 60 Hz) may lead to human health effects such as childhood leukemia or brain tumors. In a previous study investigating multiple types of cells from brain and kidney of the mouse (Acta Neuropathologica 2004; 107: 257–264), we found increased unrepaired nuclear DNA single strand breaks (nDNA SSB) only in epithelial cells of the choroid plexus in the brain using autoradiographic methods after a continuous eight-week 50 Hz magnetic field (MF) exposure of adult mice with flux density of 1.5 mT.

Methods

In the present study we tested the hypothesis that MF exposure with lower flux densities (0.1 mT, i.e., the actual exposure limit for the population in most European countries, and 1.0 mT) shows similar results to those in the previous study. Experiments and data analysis were carried out in a similar way as in our previous study.

Results

Continuous eight-week 50 Hz MF exposure with 0.1 mT or 1.0 mT did not result in increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice. MF exposure with 1.0 mT led to reduced unscheduled DNA synthesis (UDS) in epithelial cells in the choroid plexus of the fourth ventricle in the brain (EC-CP) and epithelial cells of the cortical collecting duct in the kidney, as well as to reduced mtDNA synthesis in neurons of the caudate nucleus in the brain and in EC-CP.

Conclusion

No evidence was found for increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice after continuous eight-week 50 Hz magnetic field exposure with flux density of 0.1 mT or 1.0 mT.     

Facets of diazotrophy in the oxygen minimum zone waters off Peru

Nitrogen fixation, the biological reduction of dinitrogen gas (N₂) to ammonium (NH₄⁺), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N₂ fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N₂ fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N₂ fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N₂ fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO₂⁻ and PO₄³⁻ are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N₂ fixation may increase in the future.     

Release Factor One Is Nonessential in Escherichia coli

Recoding a stop codon to an amino acid may afford orthogonal genetic systems for biosynthesizing new protein and organism properties. Although reassignment of stop codons has been found in extant organisms, a model organism is lacking to investigate the reassignment process and to direct code evolution. Complete reassignment of a stop codon is precluded by release factors (RFs), which recognize stop codons to terminate translation. Here we discovered that RF1 could be unconditionally knocked out from various Escherichia coli stains, demonstrating that the reportedly essential RF1 is generally dispensable for the E. coli species. The apparent essentiality of RF1 was found to be caused by the inefficiency of a mutant RF2 in terminating all UAA stop codons; a wild type RF2 was sufficient for RF1 knockout. The RF1-knockout strains were autonomous and unambiguously reassigned UAG to encode natural or unnatural amino acids (Uaas) at multiple sites, affording a previously unavailable model for studying code evolution and a unique host for exploiting Uaas to evolve new biological functions.     

Dendritic position is a major determinant of presynaptic strength

Different regulatory principles influence synaptic coupling between neurons, including positional principles. In dendrites of pyramidal neurons, postsynaptic sensitivity depends on synapse location, with distal synapses having the highest gain. In this paper, we investigate whether similar rules exist for presynaptic terminals in mixed networks of pyramidal and dentate gyrus (DG) neurons. Unexpectedly, distal synapses had the lowest staining intensities for vesicular proteins vGlut, vGAT, Synaptotagmin, and VAMP and for many nonvesicular proteins, including Bassoon, Munc18, and Syntaxin. Concomitantly, distal synapses displayed less vesicle release upon stimulation. This dependence of presynaptic strength on dendritic position persisted after chronically blocking action potential firing and postsynaptic receptors but was markedly reduced on DG dendrites compared with pyramidal dendrites. These data reveal a novel rule, independent of neuronal activity, which regulates presynaptic strength according to dendritic position, with the strongest terminals closest to the soma. This gradient is opposite to postsynaptic gradients observed in pyramidal dendrites, and different cell types apply this rule to a different extent.     

Cutting edge: microRNA regulation of macrophage fusion into multinucleated giant cells

Cellular fusion of macrophages into multinucleated giant cells is a distinguishing feature of the granulomatous response to inflammation, infection, and foreign bodies (Kawai and Akira. 2011. Immunity 34: 637-650). We observed a marked increase in fusion of macrophages genetically deficient in Dicer, an enzyme required for canonical microRNA (miRNA) biogenesis. Gene expression profiling of miRNA-deficient macrophages revealed an upregulation of the IL-4-responsive fusion protein Tm7sf4, and analyses identified miR-7a-1 as a negative regulator of macrophage fusion, functioning by directly targeting Tm7sf4 mRNA. miR-7a-1 is itself an IL-4-responsive gene in macrophages, suggesting feedback control of cellular fusion. Collectively, these data indicate that miR-7a-1 functions to regulate IL-4-directed multinucleated giant cell formation.     

A Holocene environmental record reflecting vegetation, climate, and fire variability at the Páramo of Quimsacocha, southwestern Ecuadorian Andes

We reconstructed the palaeoenvironmental conditions of the last ca. 8,000 years in the Tres Lagunas region of the Quimsacocha volcanic basin (ca. 3,800 m a.s.l.) in the southwestern Ecuadorian Andes. By means of a pollen and charcoal record, we analysed vegetation, fire, and climate history of this area, which is sensitive to climatic changes of both the Pacific as well as of the eastern Andes and Amazon region. Sediment deposits, pronounced increases of pollen and charcoal concentrations, and pollen taxa reflect warmer and drier conditions in the early to mid-Holocene (~8000 to 3900 cal. b.p.). During the late Holocene (2250 to −57 cal. b.p.), five warm and cold phases occurred at Quimsacocha. The most prominent cold phase possibly corresponds to the globally recognized Little Ice Age (LIA; ~600 to 100 cal. b.p.). The cold phase signal at Quimsacocha was characterized by a higher abundance of Poaceae, Iso?tes and Gentianella, which are favoured by cold and moist conditions. Frequent charcoal particles can be recorded since the early to mid-Holocene (~7600 b.p.). The high Andean tree species Polylepis underwent several phases of degradation and re-establishment in the basin, which could indicate the use of fire by pre-Columbian settlers to enhance the growth of preferred herb species. The Tres Lagunas record suggests that human populations have been influencing the environment around Quimsacocha since the last ca. 8,000 years.