The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.     

Freeware tool for analysing numbers and sizes of cell colonies

Radiation and Environmental Biophysics - The clonogenic cell survival assay is a basic method to study the cytotoxic effect of radiation and chemical toxins. In large experimental setups, counting...     

Enhancing performance in anaerobic high-solids stratified bed digesters by straw bed implementation

Anaerobic high-solids single-stage stratified bed digesters have been found to be simple and flexible design candidates for small-scale reactors located in medium- to low-technology environments. In the present study, wheat straw was used as the starter material for the stratified bed. Upon green mass feeding, the anaerobically stabilised straw bed functioned both as a biofilm support and as a particulate filter. It enabled a direct onset of 7 kg VSm(-3) batch loads, added twice a week, and permitted a low but consistent bed permeability during feeding at an average superficial flow velocity of 1 m d(-1) to be achieved. Fed-batch tests with sugar beet tops in pilot- and laboratory-scale setups at an average loading rate of 2 kg VSm(-3) d(-1) resulted in average biogas production rates of 1.2-1.4 m3 m(-3) d(-1) and methane yields of 0.31-0.36 m3 kg(-1) VS(added). At the end of the laboratory-scale feeding trial, the 200 day old straw bed had compacted to 50% of its initial volume, without any negative effects on performance being detectable.     

Effects of topology, length, and charge on the activity of a kininogen-derived peptide on lipid membranes and bacteria

Effects of topology, length, and charge on peptide interactions with lipid bilayers was investigated for variants of the human kininogen-derived peptide HKH20 (HKHGHGHGKHKNKGKKNGKH) by ellipsometry, CD, fluorescence spectroscopy, and z-potential measurements. The peptides display primarily random coil conformation in buffer and at lipid bilayers, and their lipid interaction is dominated by electrostatics, the latter evidenced by higher peptide adsorption and resulting membrane rupture for an anionic than for a zwitterionic membrane, as well as by strongly reduced adsorption and membrane rupture at high ionic strength. At sufficiently high peptide charge density, however, electrostatic interactions contribute to reducing the peptide adsorption and membrane defect formation. Truncating HKH20 into overlapping 10 amino acid peptides resulted in essentially eliminated membrane rupture and in a reduced amount peptide charges pinned at the lipid bilayer. Finally, cyclic HKH20 was found to be less efficient than the linear peptide in causing liposome rupture, partly due to a lower adsorption. Analogous results were found regarding bactericidal effects.     

Biological effectiveness of very high gamma dose rate and its implication for radiological protection

Radiation and Environmental Biophysics - Many experimental studies are carried out to compare biological effectiveness of high dose rate (HDR) with that of low dose rate (LDR). The rational for...     

Campylobacter jejuni Colonization in Wild Birds: Results from an Infection Experiment

Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations.     

Differential DNA methylation in purified human blood cells: implications for cell lineage and studies on disease susceptibility

Methylation of cytosines at CpG sites is a common epigenetic DNA modification that can be measured by a large number of methods, now even in a genome-wide manner for hundreds of thousands of sites. The application of DNA methylation analysis is becoming widely popular in complex disorders, for example, to understand part of the "missing heritability". The DNA samples most readily available for methylation studies are derived from whole blood. However, blood consists of many functionally and developmentally distinct cell populations in varying proportions. We studied whether such variation might affect the interpretation of methylation studies based on whole blood DNA. We found in healthy male blood donors there is important variation in the methylation profiles of whole blood, mononuclear cells, granulocytes, and cells from seven selected purified lineages. CpG methylation between mononuclear cells and granulocytes differed for 22% of the 8252 probes covering the selected 343 genes implicated in immune-related disorders by genome-wide association studies, and at least one probe was differentially methylated for 85% of the genes, indicating that whole blood methylation results might be unintelligible. For individual genes, even if the overall methylation patterns might appear similar, a few CpG sites in the regulatory regions may have opposite methylation patterns (i.e., hypo/hyper) in the main blood cell types. We conclude that interpretation of whole blood methylation profiles should be performed with great caution and for any differences implicated in a disorder, the differences resulting from varying proportions of white blood cell types should be considered.     

Boreal Riparian Vegetation Under Climate Change

Riparian zones in boreal areas such as humid landscapes on minerogenic soils are characterized by diverse, productive, and dynamic vegetation which will rapidly react to climate change. Climate-change models predict that in most parts of the boreal region these zones will be affected by various combinations of increased temperature, less seasonal variation in runoff, increased average discharge, changes in groundwater supply, and a more dynamic ice regime. Increasing temperatures will favor invasion of exotic species whereas species losses are likely to be minor. The hydrologic changes will cause a narrowing of the riparian zone and, therefore, locally reduce species richness whereas effects on primary production are more difficult to predict. More shifts between freezing and thawing during winter will lead to increased dynamics of ice formation and ice disturbance, potentially fostering a more dynamic and species-rich riparian vegetation. Restoration measures that increase water retention and shade, and that reduce habitats for exotic plant species adjacent to rivers can be applied especially in streams and rivers that have been channelized or deprived of their riparian forest to reduce the effects of climate change on riparian ecosystems.