Adrenodoxin--a versatile ferredoxin

Mammalian adrenodoxin (Adx) has been known for many years as an essential electron mediator in mitochondrial cytochrome P450 systems. Because of its ability to support several cytochrome P450 enzymes, it is involved not only in adrenal steroid hormone biosynthesis but also in vitamin D and bile acid metabolism. Recently, Adx is increasingly gaining attention because of its potential for pharmaceutical industry and biotechnology. With human cytochromes P450 becoming important drug targets, suitable Adx-based screening systems have to be developed to test putative new drugs. Moreover, in artificial systems, Adx has been shown to functionally interact with diverse bacterial cytochromes P450 catalyzing a variety of chemically interesting reactions. Putative biotechnological applications of such Adx-containing reconstituted systems are discussed.     

Witnessing extinction: Population genetics of the last European Rollers (Coracias garrulus) in Austria and a first phylogeographic analysis of the species across its distribution range

Due to broad‐scale habitat loss, European Rollers (Coracias garrulus) have been decreasing in numbers rapidly during the 20th century in parts of their European distribution range. In Austria, as of 2017, only a completely isolated relict population of two breeding pairs and a few non‐breeders remained in Styria compared to about 270 pairs in the 1950s. In 2018, no breeders have been recorded. Since 2002, all nestlings and adult birds in Austria have been ringed. Given the small census size, combined with lack of immigration from other populations, genetic depletion seems likely. In the present study, mitochondrial control region sequence and microsatellite data based on blood samples of nestlings from recent years were collected and compared with museum samples from historical times (when Rollers were more common and widespread in Austria) and with birds across the distribution range to arrive at a first preliminary phylogeographic dataset for the species. The mitochondrial DNA showed a decrease in variation over time in Austria, eventually reaching monomorphism, while genetic diversity of 10 microsatellite loci was higher than expected and a change in genetic structuring through time was observed. These results indicate drift effects in this relict European Roller population caused by the fast population breakdown and small population size. Our phylogeographic analysis indicates a division into a European and an Asian group, roughly (but not exactly) in accordance with the two subspecies C. garrulus garrulus (Europe) and Coracias garrulus semenowi (Asia). The lack of substructuring in the European group along with the results from nuclear DNA markers show the Austrian Rollers to be part of a formerly continuous population and opens the way to restocking the present relict population with birds from Eastern Europe (“genetic rescue”).     

A Virtual Infection Model Quantifies Innate Effector Mechanisms and Candida albicans Immune Escape in Human Blood

Candida albicans bloodstream infection is increasingly frequent and can result in disseminated candidiasis associated with high mortality rates. To analyze the innate immune response against C. albicans, fungal cells were added to human whole-blood samples. After inoculation, C. albicans started to filament and predominantly associate with neutrophils, whereas only a minority of fungal cells became attached to monocytes. While many parameters of host-pathogen interaction were accessible to direct experimental quantification in the whole-blood infection assay, others were not. To overcome these limitations, we generated a virtual infection model that allowed detailed and quantitative predictions on the dynamics of host-pathogen interaction. Experimental time-resolved data were simulated using a state-based modeling approach combined with the Monte Carlo method of simulated annealing to obtain quantitative predictions on a priori unknown transition rates and to identify the main axis of antifungal immunity. Results clearly demonstrated a predominant role of neutrophils, mediated by phagocytosis and intracellular killing as well as the release of antifungal effector molecules upon activation, resulting in extracellular fungicidal activity. Both mechanisms together account for almost of C. albicans killing, clearly proving that beside being present in larger numbers than other leukocytes, neutrophils functionally dominate the immune response against C. albicans in human blood. A fraction of C. albicans cells escaped phagocytosis and remained extracellular and viable for up to four hours. This immune escape was independent of filamentation and fungal activity and not linked to exhaustion or inactivation of innate immune cells. The occurrence of C. albicans cells being resistant against phagocytosis may account for the high proportion of dissemination in C. albicans bloodstream infection. Taken together, iterative experiment–model–experiment cycles allowed quantitative analyses of the interplay between host and pathogen in a complex environment like human blood.     

Adhesion energy of receptor-mediated interaction measured by elastic deformation

We investigated the role of receptor binding affinity in surface adhesion. A sensitive technique was developed to measure the surface energy of receptor-mediated adhesion. The experimental system involved a functionalized elastic agarose bead resting on a functionalized glass coverslip. Attractive intersurface forces pulled the two surfaces together, deforming the bead to produce an enlarged contact area. The Johnson-Kendall-Roberts (JKR) model was used to relate the surface energy of the interaction to the elasticity of the bead and the area of contact. The surface energies for different combinations of modified surfaces in solution were obtained from reflection interference contrast microscopy (RICM) measurements of the contact area formed by the bead and the coverslip. Studies with surfaces functionalized with ligand-receptor pairs showed that the relationship between surface energy and the association constant of the ligand binding has two regimes. At low binding affinity, surface energy increased linearly with the association constant, while surface energy increased logarithmically with the association constant in the high affinity regime.     

Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy

Tyrosine radicals localized in the M2 subunits of ribonucleotide reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-hemoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of ribonucleotide reductase in intact ascites tumour cells.     

Detection of coronary microembolization by Doppler ultrasound in patients with stable angina pectoris during percutaneous coronary interventions under an adjunctive antithrombotic therapy with abciximab: design and rationale of the High Intensity Transient Signals ReoPro (HITS-RP) study

Background

Embolization of atherosclerotic debris from the rupture of a vulnerable atherosclerotic plaque occurs iatrogenically during percutaneous coronary interventions (PCI) and can induce myocardial necrosis. These microembolizations are detected as high intensity transient signals (HITS) using intracoronary Doppler technology.

Presentation of the hypothesis

In the presented study we will test if abciximab (ReoPro?) infusion reduces high intensity transient signals in patients with stable angina pectoris undergoing PCI in comparison to standard therapy alone.

Testing the hypothesis

The High Intensity Transient Signals ReoPro? (HITS-RP) study will enroll 60 patients. It is a prospective, single center, randomized, double-blinded, controlled trial. The study is designed to compare the efficacy of intravenous abciximab administration for reduction of microembolization during elective PCI. Patients will be randomized in a 1:1 fashion to abciximab or placebo infusion. The primary end point of the HITS-RP-Study is the number of HITS during PCI measured by intracoronary Doppler wire. Secondary endpoints are bleeding complications, elevation of cardiac biomarkers or ECG changes after percutaneous coronary interventions, changes in coronary flow velocity reserve, hs-CRP elevation, any major adverse cardio-vascular event during one month follow-up.

Implications of the hypothesis

The HITS-RP-Study addresses important questions regarding the efficacy of intravenous abciximab administration in reducing microembolization and periprocedural complications in stable angina pectoris patients undergoing PCI.

Trial registration

The trial is registered under http://www.drks-neu.uniklinik-freiburg.de/drks_web/:DRKS00000603.     

Physicochemical and morphological characterization of two small polluted streams in southwest Germany

As a supporting component of the VALIMARproject, physicochemical investigations wereconducted monthly from 1995 to 1999 at theKrähenbach/Aich stream system (two samplingsites) and at the Körsch stream (sixsampling sites). Several physicochemicalparameters were analysed continuously bydataloggers during the entire sampling period.Moreover, a selection of the most importantmorphological parameters of the stream wererecorded in 1998.The results of these physicochemicalinvestigations demonstrated that theKrähenbach/Aich system could becharacterized as a natural rhithral submontanecarbonate stream system that is welloxygenated. Low temporal variations in pH,conductivity and chloride occurred at allsampling sites in the Krähenbach whichcoincided with the geological conditions atthis test stream. Because of relatively lowconcentrations of organic and inorganiccompounds during the entire study period thesites AB and AC in the Krähenbach/Aichsystem were classified as slightly polluted.The morphological assessment of the streamindicated a moderately affected status atboth of these streams.Physicochemical and morphologicalinvestigations along the Körsch streamsystem (KA to KE including the tributary streamSulzbach) indicated a highly affected rhithralsubmontane carbonate system characterized byhigh water velocity, flow rate and dissolvedoxygen. The Körsch, however, differssignificantly with respect to watertemperature, conductivity, pH, BOD₅,nutrients, and chloride concentrations from theKrähenbach. As a result of differentanthropogenic inputs (e.g. several sewageplants, agriculture activities and waste waterdumping) the Körsch sites KA to KD wereclassified as critically to heavilypolluted. The morphological assessmentindicated a clearly affected to damagedstatus. The upstream site KE could becharacterized as slightly polluted and itsmorphological structure as moderatelyaffected.