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A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3.
From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.
C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli
(2010)
Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477.
This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in KM = 0.23 ± 0.01 mM and kcat = 167.5 ± 3.6 s⁻¹. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.
Animal experiments and preliminary results in humans have indicated alterations of hippocampal muscarinic acetylcholine receptors (mAChR) in temporal lobe epilepsy. Patients with temporal lobe epilepsy often present with a reduction in hippocampal volume. The aim of this study was to investigate the influence of hippocampal atrophy on the quantification of mAChR with single photon emission tomography (SPET) in patients with temporal lobe epilepsy. Cerebral uptake of the muscarinic cholinergic antagonist [123I]4-iododexetimide (IDex) was investigated by SPET in patients suffering from temporal lobe epilepsy of unilateral (n=6) or predominantly unilateral (n=1) onset. Regions of interest were drawn on co-registered magnetic resonance images. Hippocampal volume was determined in these regions and was used to correct the SPET results for partial volume effects. A ratio of hippocampal IDex binding on the affected side to that on the unaffected side was used to detect changes in muscarinic cholinergic receptor density. Before partial volume correction a decrease in hippocampal IDex binding on the focus side was found in each patient. After partial volume no convincing differences remained. Our results indicate that the reduction in hippocampal IDex binding in patients with epilepsy is due to a decrease in hippocampal volume rather than to a decrease in receptor concentration.
Single-photon emission tomography (SPET) with the amino acid analogue l-3-[123I]iodo-α-methyl tyrosine (IMT) is helpful in the diagnosis and monitoring of cerebral gliomas. Radiolabelled amino acids seem to reflect tumour infiltration more specifically than conventional methods like magnetic resonance imaging and computed tomography. Automatic tumour delineation based on maximal tumour uptake may cause an overestimation of mean tumour uptake and an underestimation of tumour extension in tumours with circumscribed peaks. The aim of this study was to develop a program for tumour delineation and calculation of mean tumour uptake which takes into account the mean background activity and is thus optimised to the problem of tumour definition in IMT SPET. Using the frequency distribution of pixel intensities of the tomograms a program was developed which automatically detects a reference brain region and draws an isocontour region around the tumour taking into account mean brain radioactivity. Tumour area and tumour/brain ratios were calculated. A three-compartment phantom was simulated to test the program. The program was applied to IMT SPET studies of 20 patients with cerebral gliomas and was compared to the results of manual analysis by three different investigators. Activity ratios and chamber extension of the phantom were correctly calculated by the automatic analysis. A method based on image maxima alone failed to determine chamber extension correctly. Manual region of interest analysis in patient studies resulted in a mean inter-observer standard deviation of 8.7%±6.1% (range 2.7%–25.0%). The mean value of the results of the manual analysis showed a significant correlation to the results of the automatic analysis (r = 0.91, P<0.0001 for the uptake ratio; r = 0.87, P<0.0001 for the tumour area). We conclude that the algorithm proposed simplifies the calculation of uptake ratios and may be used for observer-independent evaluation of IMT SPET studies. Three-dimensional tumour recognition and transfer to co-registered morphological images based on this program may be useful for the planning of surgical and radiation treatment.
Unravelling the factors determining the allocation of carbon to various plant organs is one of the great challenges of modern plant biology. Studying allocation under close to natural conditions requires non-invasive methods, which are now becoming available for measuring plants on a par with those developed for humans. By combining magnetic resonance imaging (MRI) and positron emission tomography (PET), we investigated three contrasting root/shoot systems growing in sand or soil, with respect to their structures, transport routes and the translocation dynamics of recently fixed photoassimilates labelled with the short-lived radioactive carbon isotope 11C. Storage organs of sugar beet (Beta vulgaris) and radish plants (Raphanus sativus) were assessed using MRI, providing images of the internal structures of the organs with high spatial resolution, and while species-specific transport sectoralities, properties of assimilate allocation and unloading characteristics were measured using PET. Growth and carbon allocation within complex root systems were monitored in maize plants (Zea mays), and the results may be used to identify factors affecting root growth in natural substrates or in competition with roots of other plants. MRI–PET co-registration opens the door for non-invasive analysis of plant structures and transport processes that may change in response to genomic, developmental or environmental challenges. It is our aim to make the methods applicable for quantitative analyses of plant traits in phenotyping as well as in understanding the dynamics of key processes that are essential to plant performance.
Within ESA's Cosmic Vision 2015-2025 plan, a mission to explore the Saturnian System, with special emphasis on its two moons Titan and Enceladus, was selected for study, termed TANDEM (Titan and Enceladus Mission). In this paper, we describe an optimized mission design for a TANDEM-derived solar electric propulsion (SEP) mission. We have chosen the SEP mission scenario for the interplanetary transfer of the TANDEM spacecraft because all feasible gravity assist sequences for a chemical transfer between 2015 and 2025 result in long flight times of about nine years. Our SEP system is based on the German RIT ion engine. For our optimized mission design, we have extensively explored the SEP parameter space (specific impulse, thrust level, power level) and have calculated an optimal interplanetary trajectory for each setting. In contrast to the original TANDEM mission concept, which intends to use two launch vehicles and an all-chemical transfer, our SEP mission design requires only a single Ariane 5 ECA launch for the same payload mass. Without gravity assist, it yields a faster and more flexible transfer with a fight time of less than seven years, and an increased payload ratio. Our mission design proves thereby the capability of SEP even for missions into the outer solar system.
Successful bone sawing requires a high level of skill and experience, which could be gained by the use of Virtual Reality-based simulators. A key aspect of these medical simulators is realistic force feedback. The aim of this paper is to model the bone sawing process in order to develop a valid training simulator for the bilateral sagittal split osteotomy, the most often applied corrective surgery in case of a malposition of the mandible. Bone samples from a human cadaveric mandible were tested using a designed experimental system. Image processing and statistical analysis were used for the selection of four models for the bone sawing process. The results revealed a polynomial dependency between the material removal rate and the applied force. Differences between the three segments of the osteotomy line and between the cortical and cancellous bone were highlighted.
An Interstellar – Heliopause mission using a combination of solar/radioisotope electric propulsion
(2011)
There is common agreement within the scientific community that in order to understand our local galactic environment it will be necessary to send a spacecraft into the region beyond the solar wind termination shock. Considering distances of 200 AU for a new mission, one needs a spacecraft travelling at a speed of close to 10 AU/yr in order to keep the mission duration in the range of less than 25 yrs, a transfer time postulated by ESA.Two propulsion options for the mission have been proposed and discussed so far: the solar sail propulsion and the ballistic/radioisotope electric propulsion. As a further alternative, we here investigate a combination of solar-electric propulsion and radioisotope-electric propulsion. The solar-electric propulsion stage consists of six 22 cm diameter “RIT-22”ion thrusters working with a high specific impulse of 7377 s corresponding to a positive grid voltage of 5 kV. Solar power of 53 kW BOM is provided by a light-weight solar array. The REP-stage consists of four space-proven 10 cm diameter “RIT-10” ion thrusters that will be operating one after the other for 9 yrs in total. Four advanced radioisotope generators provide 648 W at BOM. The scientific instrument package is oriented at earlier studies. For its mass and electric power requirement 35 kg and 35 W are assessed, respectively. Optimized trajectory calculations, treated in a separate contribution, are based on our “InTrance” method.The program yields a burn out of the REP stage in a distance of 79.6 AU for a usage of 154 kg of Xe propellant. With a C3 = 45,1 (km/s)2 a heliocentric probe velocity of 10 AU/yr is reached at this distance, provided a close Jupiter gravity assist adds a velocity increment of 2.7 AU/yr. A transfer time of 23.8 yrs results for this scenario requiring about 450 kg Xe for the SEP stage, jettisoned at 3 AU. We interpret the SEP/REP propulsion as a competing alternative to solar sail and ballistic/REP propulsion. Omiting a Jupiter fly-by even allows more launch flexibility, leaving the mission duration in the range of the ESA specification.
Solar-electric propulsion (SEP) is superior with
respect to payload capacity, flight time and
flexible launch window to the conventional
interplanetary transfer method using chemical
propulsion combined with gravity assists. This fact
results from the large exhaust velocities of electric
low–thrust propulsion and is favourable also for
missions to the giant planets, Kuiper-belt objects
and even for a heliopause probe (IHP) as shown in
three studies by the authors funded by DLR. They
dealt with a lander for Europa and a sample return
mission from a mainbelt asteroid [1], with the
TANDEM mission [2]; the third recent one
investigates electric propulsion for the transfer to
the edge of the solar system.
All studies are based on triple-junction solar arrays,
on rf-ion thrusters of the qualified RIT-22 type and
they use the intelligent trajectory optimization
program InTrance [3].