Activation of Humoral Immunity during the Pathogenesis of Experimental Chronic Lung Allograft Dysfunction

Alloreactive and autoreactive antibodies have been associated with the development of chronic lung allograft dysfunction (CLAD), but their pathogenic role is disputed. Orthotopic left lung transplantation was performed in the Fischer-344 to Lewis rat strain combination followed by the application of ciclosporine for 10 days. Four weeks after transplantation, lipopolysaccharide (LPS) was instilled into the trachea. Lungs were harvested before (postoperative day 28) and after LPS application (postoperative days 29, 33, 40, and 90) for histopathological, immunohistochemical, and Western blot analyses. Recipient serum was collected to investigate circulating antibodies. Lung allografts were more strongly infiltrated by B cells and deposits of immunoglobulin G and M were more prominent in allografts compared to right native lungs or isografts and increased in response to LPS instillation. LPS induced the secretion of autoreactive antibodies into the circulation of allograft and isograft recipients, while alloreactive antibodies were only rarely detected. Infiltration of B cells and accumulation of immunoglobulin, which is observed in allografts treated with LPS but not isografts or native lungs, might contribute to the pathogenesis of experimental CLAD. However, the LPS-induced appearance of circulating autoreactive antibodies does not seem to be related to CLAD, because it is observed in both, isograft and allograft recipients.     

Trifluoroselenomethionine: A New Unnatural Amino Acid

Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N‐(tert‐butoxycarbonyl)‐l ‐aspartic acid tert‐butyl ester. TFSeM shows enhanced methioninase‐induced cytotoxicity, relative to selenomethionine (SeM), toward HCT‐116 cells derived from human colon cancer. Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. Nonetheless, when two different variants of the protein GB1 were expressed in an Escherichia coli methionine auxotroph cell line in the presence of TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85 % of the proteins contained SeM residues, even though no SeM had been added, thus implying loss of the trifluoromethyl group from TFSeM. The transformation of TFSeM into SeM is enzymatically catalyzed by E. coli extracts, but TFSeM is not a substrate of E. coli methionine adenosyltransferase.     

A genomic screening approach to the structure-guided identification of drug candidates from natural sources

The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence-guided genetic screening strategy. In this "genome mining" approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties.     

Isolation, Identification, Synthesis, and Field Evaluation of the Sex Pheromone of the Brazilian Population of Spodoptera frugiperda

Several studies have shown intraspecific geographical variation in the composition of sex pheromones. Pheromone lures from North America and Europe were not effective against the fall armyworm Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) in Brazil, so we examined the composition of the sex pheromone produced by females from Brazilian populations. Virgin female gland extracts contained (Z)-7-dodecenyl acetate (Z7-12:Ac), (E)-7-dodecenyl acetate (E7-12:Ac), dodecyl acetate, (Z)-9-dodecenyl acetate, (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z)-10-tetradecenyl acetate, tetradecyl acetate/(Z)-11-tetradecenyl acetate (Z11-16:Ac), and (Z)-11-hexadecenyl acetate. The relative proportions of each acetate were 0.8:1.2:0.6:traces:82.8:0.3:1.5:12.9, respectively. This is the first time that E7-12:Ac has been reported from the pheromone gland of S. frugiperda. Only three compounds, Z9-14:Ac, Z7-12:Ac, and E7-12:Ac, elicited antennal responses, and there were no differences in catch between traps baited with either Z7-12:Ac + Z9-14:Ac or Z7-12:Ac + Z9-14:Ac + Z11-16:Ac blends. However, the Z7-12:Ac + Z9-14:Ac + E7-12:Ac blend was significantly better than Z7-12:Ac + Z9-14:Ac, indicating that E7-12:Ac is an active component in the sex pheromone of the Brazilian populations of S. frugiperda.     

High resolution 3D gas-jet characterization

We present a tomographic characterization of gas jets employed for high-intensity laser-plasma interaction experiments where the shape can be non-symmetrically. With a Mach-Zehnder interferometer we measured the phase shift for different directions through the neutral density distribution of the gas jet. From the recorded interferograms it is possible to retrieve 3-dimensional neutral density distributions by tomographic reconstruction based on the filtered back projections. We report on criteria for the smallest number of recorded interferograms as well as a comparison with the widely used phase retrieval based on an Abel inversion. As an example for the performance of our approach, we present the characterization of nozzles with rectangular openings or gas jets with shock waves. With our setup we obtained a spatial resolution of less than 60?μm for an Argon density as low as 2 × 10(17) cm(-3).     

Einfluß von Schwermetallionen in Trinkwasser auf die Aktivität des AChE-Biosensors

Influence of Heavy Metal Ions in Drinking Water on the Activity of the AChE-Biosensor Because of the specific inhibition of the enzyme acetylcholinesterase (AChE) by organophosphoric ester, -thioester and N-methylcarbamates an acetylcholinesterase biosensor can be used to screen drinking water for the presence of compounds used as insecticides. The presence of heavy metal ions may also lead to a decrease in catalytic activity of the enzyme, and in this way a misinterpretation of the results of the screening is possible. In this paper the effect of lead, iron, copper and zinc ions on the activity was examined, with the view to the concentration limit given by the decree for drinking water in the Federal Republic of Germany. Zinc and copper ions in drinking water produce a detectable inhibition (3 mg/L copper produced an inhibition of 10 to 20%, 5 mg/L zinc an inhibition of 10%), therefore the presence of copper, lead and other heavy metal ions not tested in drinking water may produce wrong results in pesticide screening. The coimmobilization of albumin and enzyme was successfull in eliminating the inhibition caused by heavy metal ions. Using this membrane for a 3 mg/L copper solution an inhibition of only 0.3% higher was measured and for a 5 mg/L zinc solution an inhibition of 2.3% lower than the inhibition of TRIS-buffer itself. This results were in the range of the standard deviation, which means that no inhibition could be measured with this metal ion solution using albumin containing AChE-membranes. The use of this modified membrane in real drinking water samples shows clearly lower inhibition values than that of membranes without albumin.     

Hybrid separations combining distillation and organic solvent nanofiltration for separation of wide boiling mixtures

Membrane assisted hybrid separations offer tremendous potential for process intensification which aims at increasing resource efficiency as well as decreasing operating and capital costs. Design of such processes is challenging due to large number of degrees of freedom but also due to large experimental effort necessary for membrane screening and for characterising membranes in whole operating range. To address these issues, this paper elaborates a four-step design method for combination of organic solvent nanofiltration (OSN) and distillation in a hybrid separation of wide boiling mixtures. The design method is applied in a case study which is the separation of small amounts of heavy boiler from a mixture containing a mid- and a light-boiler. In the first step, different process options are generated based on heuristics and engineering judgement and screened for feasibility. In the second step, the options are evaluated based on quantitative metrics using rigorous models. In this step the unknown key parameters are identified, and their influences on the process performance are quantified in a detailed a priori process analysis. If hybrid separations with OSN show to be promising when compared to stand-alone distillation, experiments are conducted to (i) identify the best membrane for the operating window in which the hybrid process operates and (ii) to perform model validation and parameterisation in the third step. In the last (fourth) step, an optimisation is performed to identify the best (cost optimal) process using the experimental data gained in step three.     

Configurable Low‐Cost Plotter Device for Fabrication of Multi‐Color Sub‐Cellular Scale Microarrays

The construction and operation of a low‐cost plotter for fabrication of microarrays for multiplexed single‐cell analyses is reported. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab‐View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15 000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 cm² using up to three different oligonucleotides. Typical feature sizes are 5 μm diameter with a pitch of 15 μm, leading to densities of up to 10⁴–10⁵ spots/mm². The fabricated DNA microarrays are used to produce sub‐cellular scale arrays of bioactive epidermal growth factor peptides by means of DNA‐directed immobilization. The suitability of these biochips for cell biological studies is demonstrated by specific recruitment, concentration, and activation of EGF receptors within the plasma membrane of adherent living cells. This work illustrates that the presented plotter gives access to bio‐functionalized arrays usable for fundamental research in cell biology, such as the manipulation of signal pathways in living cells at subcellular resolution.