Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10³–10⁴ V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strenght it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yields a mean value of about 1.6 V for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 °C as shown by incorporation of ¹³¹I-labeled albumin and repeated dielectric breakdown.     

Viral control of bacterial biodiversity – evidence from a nutrient-enriched marine mesocosm experiment

We demonstrate here results showing that bottom-up and top-down control mechanisms can operate simultaneously and in concert in marine microbial food webs, controlling prokaryote diversity by a combination of viral lysis and substrate limitation. Models in microbial ecology predict that a shift in the type of bacterial growth rate limitation is expected to have a major effect on species composition within the community of bacterial hosts, with a subsequent shift in the composition of the viral community. Only moderate effects would, however, be expected in the absolute number of coexisting virus–host pairs. We investigated these relationships in nutrient-manipulated systems, under simulated in situ conditions. There was a strong correlation in the clustering of the viral and bacterial community data supporting the existence of an important link between the bacterial and viral communities. As predicted, the total number of viral populations was the same in all treatments, while the composition of the viral community varied. Our results support the theoretical prediction that there is one control mechanism for the number of niches for coexisting virus–host pairs (top-down control), and another mechanism that controls which virus–host pairs occupy these niches (bottom-up control).     

Diversity and abundance of freshwater Actinobacteria along environmental gradients in the brackish northern Baltic Sea

Actinobacteria are highly abundant in pelagic freshwater habitats and also occur in estuarine environments such as the Baltic Sea. Because of gradients in salinity and other environmental variables estuaries offer natural systems for examining factors that determine Actinobacteria distribution. We studied abundance and community structure of Bacteria and Actinobacteria along two transects in the northern Baltic Sea. Quantitative (CARD-FISH) and qualitative (DGGE and clone libraries) analyses of community composition were compared with environmental parameters. Actinobacteria accounted for 22–27% of all bacteria and the abundance changed with temperature. Analysis of 549 actinobacterial 16S rRNA sequences from four clone libraries revealed a dominance of the freshwater clusters ac I and ac IV, and two new subclusters ( ac I-B scB-5 and ac IV-E) were assigned. Whereas ac I was present at all stations, occurrence of ac II and ac IV differed between stations and was related to dissolved organic carbon (DOC) and chlorophyll a (Chl a ) respectively. The prevalence of the ac I-A and ac I-B subclusters changed in relation to total phosphorus (Tot-P) and Chl a respectively. Community structure of Bacteria and Actinobacteria differed between the river station and all other stations, responding to differences in DOC, Chl a and bacterial production. In contrast, the composition of active Actinobacteria (analysis based on reversely transcribed RNA) changed in relation to salinity and Tot-P. Our study suggests an important ecological role of Actinobacteria in the brackish northern Baltic Sea. It highlights the need to address dynamics at the cluster or subcluster phylogenetic levels to gain insights into the factors regulating distribution and composition of Actinobacteria in aquatic environments.     

Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.     

Toxoplasma gondii antibodies in free-living African mammals.

Twelve species of free-living African mammals from Kenya, Tanzania, Uganda and Zambia were tested for antibodies to Toxoplasma gondii using the indirect hemagglutination test. Of 157 animals sampled, 20 (13%) were seropositive. T. gondii antibodies were detected in Burchell's zebra, (Equus burchelli), hippopotamus (Hippopotamus amphibius), African elephant (Loxodonta africana), defassa waterbuck (Kobus defassa), lion (Panthera leo), and rock hyrax (Procavia capensis), The highest titers were found in elephants, two having titers of 1:4096 and one of 1:8192. These results are discussed in relation to the maintenance of T. gondii among African wildlife.     

Dielectric Breakdown of Cell Membranes

With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 · 10⁶ V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector voltage (or the electric field strength in the orifice) depends on the membrane composition (or the intrinsic membrane potential) as revealed by measuring the critical voltage in E. coli B harvested from the logarithmic and stationary growth phases. The critical detector voltage increased by about 30% for a given volume on reaching the stationary growth phase.     

PET-compatible endothelin receptor radioligands: Synthesis and first in vitro and in vivo studies

The expression and function of endothelin (ET) receptors is abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. In this study, we prepared two [¹⁸F]-fluorinated derivatives of the non-peptide ET_A receptor antagonist PD 156707 and evaluated their ET receptor binding potencies. Ex vivo as well as in vivo biodistribution studies in mice were performed, as well as the metabolism of the radiotracer, which was examined by metabolite analysis in mice and rats. All tested derivatives of PD 156707 exhibited potent in vitro pharmacological characteristics with K_i values comparable to that of the lead compound. The biodistribution studies showed a high accumulation of the tracer in bile and intestine. In vivo we were able to show that the visualization of the heart as a major target organ with high ET_AR expression is possible.     

The Relationship between Brain Morphology and Polysomnography in Healthy Good Sleepers

BackgroundNormal sleep continuity and architecture show remarkable inter-individual variability. Previous studies suggest that brain morphology may explain inter-individual differences in sleep variables.MethodThirty-eight healthy subjects spent two consecutive nights at the sleep laboratory with polysomnographic monitoring. Furthermore, high-resolution T1-weighted MRI datasets were acquired in all participants. EEG sleep recordings were analyzed using standard sleep staging criteria and power spectral analysis. Using the FreeSurfer software for automated segmentation, 174 variables were determined representing the volume and thickness of cortical segments and the volume of subcortical brain areas. Regression analyses were performed to examine the relationship with polysomnographic and spectral EEG power variables.ResultsThe analysis did not provide any support for the a-priori formulated hypotheses of an association between brain morphology and polysomnographic variables. Exploratory analyses revealed that the thickness of the left caudal anterior cingulate cortex was positively associated with EEG beta2 power (24–32 Hz) during REM sleep. The volume of the left postcentral gyrus was positively associated with periodic leg movements during sleep (PLMS).ConclusionsThe function of the anterior cingulate cortex as well as EEG beta power during REM sleep have been related to dreaming and sleep-related memory consolidation, which may explain the observed correlation. Increased volumes of the postcentral gyrus may be the result of increased sensory input associated with PLMS. However, due to the exploratory nature of the corresponding analyses, these results have to be replicated before drawing firm conclusions.