High-level expression of the mycobacterial porin MspA in Escherichia coli and purification of the recombinant protein

MspA is the prototype of a new family of tetrameric porins and provides the main general diffusion pathway for hydrophilic compounds through the outer membrane of Mycobacterium smegmatis. Structural analysis was hampered by the scarce amount of pure protein. After replacement of the GC-rich codons of the mspA gene by codons optimal for high-level expression in Escherichia coli, the mature MspA protein was overproduced in E. coli. The recombinant MspA (rMspA) monomer (M(r) 20000) was purified by anion exchange and hydrophobic interaction chromatography yielding 2.6 mg pure protein per liter of culture. This exceeded the yield of the native protein 10-fold. Circular dichroism revealed that rMspA is folded in a native-like structure. rMspA assembled partially to the channel-forming tetramer both during expression in E. coli and after purification in vitro. Thus, overexpression in E. coli and chromatographic purification are key steps towards a high resolution structure of MspA.     

Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells

Immunotoxins consist of a target-cell-specific binding moiety, chemically or recombinantly linked to a cytotoxic component. A number of different immunotoxins (IT) have increasingly been evaluated for immunotherapy. Since these foreign proteins are highly immunogenic in human, we have developed recombinant IT using the human ribonuclease angiogenin. Due to their potential toxic effects on eucaryotic cells, these IT are usually expressed in bacteria. Depending on the structure, size, and sequence of the desired IT, bacterial expression can be limited and the yield after purification be unsatisfactory. Therefore, the expression of IT in eucaryotic cells could provide a promising alternative. For this purpose we genetically fused the anti-CD30 single-chain variable fragment (scFv) Ki4 to the N- and C-termini of recombinant angiogenin. Both IT possess leader sequences, which mediate their secretion into the cell culture supernatant. Using a bicistronic mRNA the IT were simultaneously expressed together with enhanced green fluorescent protein (EGFP). This allows direct monitoring of transfected cells. An additional plasmid encoded Zeocin resistance enhances the cultivation of transfected cells under selection pressure. Three days after transfection of 293T-cells, unpurified IT were analyzed by flow cytometry and competitive cell proliferation assays. This is the first report on the use of eucaryotic cells for the secretion of functionally active IT with a human effector domain.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Discounting and the environment should current impacts be weighted differently than impacts harming future generations?

Background  In Life-Cycle Assessment (LCA), decision makers are often faced with tradeoffs between current and future impacts. One typical example is waste incineration, where immediate emissions to the air from the incineration process have to be weighted against future emissions of slag landfills. Long-term impacts are either completely taken into account or they are entirely disregarded in case of a temporal cut-off. Temporal cutoffs are a special case of discounting. Objective  In this paper, discounting is defined as valuing damages differently at different points of time using a positive or negative discount rate. Apart from temporal cut-offs, discounting has rarely been applied in LCA so far. It is the goal of this paper to discuss the concept of discounting and its applicability in the context of LCA. Methods  For this purpose, we first review the arguments for discounting and its principles in economic sciences. Discounting in economics can be motivated by pure time preference, productivity of capital, diminishing marginal utility of consumption, and uncertainties. The nominal discount rate additionally includes changes in the price level. These arguments and their justification are discussed in the context of environmental impacts harming future generations. Results and Discussion  It is concluded that discounting across generations because of pure time preference contradicts fundamental ethical values and should therefore not be applied in LCA. However, it has to be acknowledged that in practice decision makers often use positive discount rates because of pure time preference — either because they might profit from imposing environmental damage on others instead of themselves or because people in the far future are not of immediate concern to them. Discounting because of the productivity of capital assumes a relationship between monetary values and environmental impact. If such a relationship is accepted, discounting could be applied. However, future generations should be compensated for the environmental damage. It is likely that they would demand a higher compensation if the real per capita income increases. As both the compensation and the discount rate are related to economic growth, the overall discount rate might be close to zero. It is shown that the overall discount rate might even be negative considering that the required compensation could increase (even to infinite) if natural assets remain scarce, whereas the utility of consumption diminishes with increasing income. Uncertainties could justify both positive and negative discount rates. Since the relationship between uncertainties and the magnitude of damage is generally not exponential, we recommend to model changes in the magnitude of damage in scenario analysis instead of considering it in discounting (which requires an exponential function of time in the case of a constant discount rate). We investigated the influence of discounting in a case study of heavy metal emissions from slag landfills. It could be shown that even small discount rates of less than 1 % lead to a significant reduction of the impact score, whereas negative discount rates inflate the results. Conclusions and Recommendations  Discounting is only applicable when temporally differentiated data is available. In some cases, such a temporal differentiation is necessary to take sound decisions, especially when long emission periods are involved. An example is the disposal of nuclear or heavy metal-containing waste. In these cases, the results might completely depend on the discount rate. This paper helps to structure arguments and thus to support the decision about whether or not discounting should be applied in an LCA.     

Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate α-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5ng of transferred protein in a single band and is thus 5–10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.     

Seasonal Community and Population Dynamics of Pelagic Bacteria and Archaea in a High Mountain Lake

The seasonal variations in community structure and cell morphology of pelagic procaryotes from a high mountain lake (Gossenköllesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and image-analyzed microscopy. Compositional changes and biomass fluctuations within the assemblage were observed both in summer and beneath the winter ice cover and are discussed in the context of physicochemical and biotic parameters. Proteobacteria of the beta subclass (beta-proteobacteria) formed a dominant fraction of the bacterioplankton (annual mean, 24% of the total counts), whereas alpha-proteobacteria were of similar relative importance only during spring (mean, 11%). Bacteria of the Cytophaga-Flavobacterium cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during most of the year, thus contributing significantly to the total microbial biomass. Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of ice cover formation, respectively. A set of oligonucleotide probes targeted to single phylotypes was constructed from 16S rRNA-encoding gene clone sequences. Three distinct populations of uncultivated microbes, affiliated with the alpha- and beta-proteobacteria, were subsequently monitored by FISH. About one-quarter of all of the beta-proteobacteria (range, 6 to 53%) could be assigned to only two phylotypes. The bacterial populations studied were annually recurrent, seasonally variable, and vertically stratified, except during the periods of lake overturn. Their variability clearly exceeded the fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.Until recently, microbial ecologist studying aquatic bacteria faced a basic dilemma: they could either measure the abundance, biomass, growth rates, activity, etc. of the “average” bacterium under in situ conditions (e.g., see reference 13), ignoring the phylogenetic and physiological diversity of microbial communities, or they could isolate and ecophysiologically characterize individual bacterial strains (e.g., see reference 36) but were then not able to tell if these microorganisms were also common in the environment. Consequently, little knowledge has been gathered about the spatial and temporal abundance fluctuations of defined phylogenetic groups and of individual bacterial species in natural habitats. Molecular biological techniques used to identify microbes in environmental samples have recently provided new tools to study bacterioplankton biodiversity (e.g., see references 1, 9, 14, 15, and 19) and the in situ abundances of bacteria and archaea that could not be adequately distinguished before (2, 4, 5, 25). Microbiologists are now in a position to potentially elucidate the biogeography (24), population dynamics, and successions (28) not only of a few morphologically conspicuous microbes but of a large number of species, most of which might still be uncharacterized.Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes selectively visualizes bacterial cells with defined phylogenetic affiliations (3, 5). Based on a rapidly growing set of 16S (and, to a lesser extend, 23S) rRNA sequence data, it is probably the phylogenetically most sophisticated (22) approach for whole-cell in situ identification. On the other hand, FISH of plankton samples can be performed with minimal laboratory requirements (16), and evaluation relies on epifluorescence microscopy, which is a standard technique of aquatic microbial ecologists, e.g., for counting (30) and sizing (33) of picoplankton. In contrast to other identification approaches, FISH largely conserves the gestalt of the targeted microorganisms, i.e., their morphologies, cell sizes (26, 34), and cellular rRNA content (7, 32). So, despite the limitations of the method (as discussed in reference 5), its potential for the identification and cytometric analysis of planktonic microbes is just about to be recognized.Recent investigations have reported that various freshwater microbial communities are dominated by bacteria which are phylogenetically affiliated with the alpha and beta subclasses of the class Proteobacteria (alpha- and beta-proteobacteria, respectively) and with members of the Cytophaga-Flavobacterium cluster (2, 6, 16, 19). These observations were based on single or short-term sampling schemes. The instantaneous community composition of the bacterioplankton, however, may not be representative for different seasons, and the typical ranges of annual community variability remain to be established.The size distribution of planktonic bacteria, and particularly the appearance of filamentous cells, has come into the focus of aquatic microbial ecology in the context of studies of predator-prey interactions. It has been shown both in the laboratory (18, 37) and in field experiments (20) that the filamentous morphotype is a phenotypic adaptation of some microbes to protistan grazing, but there are probably numerous other causes for bacteria to elongate far beyond their typical sizes (e.g., see reference 23). Threadlike bacteria have been observed throughout the year in the plankton of a hypertrophic lake (41) but were also found in midwinter in an oligotropic alpine lake (31).In earlier studies, we demonstrated FISH to be an appropriate tool for the monitoring of spatial (2) and short-term temporal (26) dynamics of different phylogenetic groups of the planktonic microbial community in a high mountain lake. Here we report on the seasonal and vertical abundance distributions of pelagic members of Bacteria and Archaea in Gossenköllesee and analysis of the community structure at different levels of taxonomic resolution. We applied published domain- and group-specific oligonucleotide probes (5) but also used the sequence information from a 16S rRNA-encoding gene (rDNA) library obtained from Gossenköllesee bacterioplankton 1 year earlier to construct specific probes targeted at individual bacterial populations. Particular attention was paid to the changes in abundance and taxonomic composition of the filamentous bacterial morphotypes which were recognized as a permanently important fraction of the planktonic procaryotes in Gossenköllesee. Additionally, we monitored the seasonal changes in the biomass size distributions of the nonfilamentous fraction of the pelagic microbial community.