Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.     

Licht- und elektronenmikroskopische Untersuchungen zur Wirkung eines Kulturfiltrates von Erwinia herbicola B 247 und von Herbicolin A auf Fusarium culmorum

Light and electron microscopical studies on the effect of a culture filtrate of Erwina herbicola B 247 and herbicolin A on Fusarium culmorum The effect of a culture filtrate of Erwinia herbicola B 247 and the antibiotic herbicolin A, respectively, on the hyphae of Fusarium culmorum was studied in vitro using light and electron microscopy. The light microscopy revealed a swelling and disruption of the hyphae tips with a release of cytoplasm. Ultrastructural investigations demonstrated the appearance of electron-dense material of a round or tubular structure in the cell wall. On its inner side, an accumulation of electron-dense material formed a spongy structure associated with the altered plasma membrane. Finally, a complete dissolution of the cell wall was observed.     

Rapid Stereoselective Hydrolysis of (+)-Cocaine in Baboon Plasma Prevents Its Uptake in the Brain: Implications for Behavioral Studies

The naturally occurring enantiomer of cocaine, (–)-cocaine, has been previously labeled with ¹¹C on the N-methyl group and used in conjunction with positron emission tomography to show that cocaine is rapidly taken up in the striata of human and baboon brain. In the present study, the behaviorally inactive (+)-cocaine was similarly labeled, with a view to its use for measuring the nonspecific binding of cocaine. No brain uptake was seen, although transport of cocaine into the brain is not expected to be stereoselective. The explanation for the lack of uptake was determined to be very rapid metabolism of (+)-cocaine in the blood. By 30s after administration of labeled (+)-cocaine, it was undetectable in plasma. In vitro studies demonstrated that (+)-cocaine is 50% debenzoylated to (+)-ecgonine methyl ester within 5 s of exposure to baboon plasma but not to washed erythrocytes. The hydrolysis of (–)-cocaine is at least 1,000 times slower. Serum butyrylcholinesterase (EC 3.1.1.8) appears to be responsible for this hydrolysis, as evidenced by its inhibition by physostigmine and catalysis by commercially available pseudocholinesterase from horse and human blood.     

Characterization of the interaction of doxorubicin with (poly)phosphoinositides in model systems. Evidence for specific interaction with phosphatidylinositol-monophosphate and -diphosphate.

The anticancer drug doxorubicin penetrates into Langmuir monolayers containing phosphoinositides. Upon binding of doxorubicin to phosphoinositide-containing SUV, its fluorescence is self-quenched due to self-association. As compared to other anionic phospholipids, as much as 2- to 3-fold larger effects were obtained with PIP and PIP2, in mixtures of these lipids with DOPC. Doxorubicin competes efficiently with the non-penetrating antibiotic neomycin for binding to PIP2. According to its penetration, specific binding of doxorubicin was half-maximal at 5-15 microM. It is likely that also in biological membranes doxorubicin binds specifically to PIP and PIP2.     

Environment versus dispersal in the assembly of western Amazonian palm communities

Aim It is a central issue in ecology and biogeography to understand what governs community assembly and the maintenance of biodiversity in tropical rain forest ecosystems. A key question is the relative importance of environmental species sorting (niche assembly) and dispersal limitation (dispersal assembly), which we investigate using a large dataset from diverse palm communities. Location Lowland rain forest, western Amazon River Basin, Peru. Methods We inventoried palm communities, registering all palm individuals and recording environmental conditions in 149 transects of 5 m × 500 m. We used ordination, Mantel tests and indicator species analysis (ISA) to assess compositional patterns, species responses to geographical location and environmental factors. Mantel tests were used to assess the relative importance of geographical distance (as a proxy for dispersal limitation) and environmental differences as possible drivers of dissimilarity in palm species composition. We repeated the Mantel tests for subsets of species that differ in traits of likely importance for habitat specialization and dispersal (height and range size). Results We found a strong relationship between compositional dissimilarity and environmental distance and a weaker but also significant relationship between compositional dissimilarity and geographical distance. Consistent with expectations, relationships with environmental and geographical distance were stronger for understorey species than for canopy species. Geographical distance had a higher correlation with compositional dissimilarity for small‐ranged species compared with large‐ranged species, whereas the opposite was true for environmental distance. The main environmental correlates were inundation and soil nutrient levels. Main conclusions The assembly of palm communities in the western Amazon appears to be driven primarily by species sorting according to hydrology and soil, but with dispersal limitation also playing an important role. The importance of environmental characteristics and geographical distance varies depending on plant height and geographical range size in agreement with functional predictions, increasing our confidence in the inferred assembly mechanisms.     

Automated precolumn fluorescence labelling by carbodiimide activation of N-acetylaspartate and N-acetylaspartylglutamate applied to an HPLC brain tissue analysis.

An automated method is described to couple carboxyl-containing metabolites to the fluorophore 2-aminoanthracene in aqueous solution (containing 75% methanol) in the presence of N,N-dicyclohexylcarbodiimide. The reaction was optimized for N-acetylaspartate (N-Ac-Asp) and N-acetylaspartylglutamate (N-Ac-Asp-Glu). The reactions occurred within 5 min at room temperature in the presence of 0.5-2 mM HCl. At concentrations of electrolytes exceeding 10 mM the coupling reaction became suboptimal. Derivatization was performed in a commercial precolumn derivatization unit. Additional tubing was needed to provide the reagents prior to reversed-phase HPLC and fluorescence detection. The assay is linear over at least three orders of magnitude; as little as 1 pmol could reproducibly be assayed in 100 micrograms wet weight brain tissue extracted with a mixture of methanol and 4 mM HCl (9:1, v/v). N-Ac-Asp and N-Ac-Asp-Glu levels in several brain regions and spinal cord were similar to those so far reported. The compounds could not be detected in peripheral tissue. The advantages, prospects and limitations of the present approach over existing methods to estimate water-soluble carboxylic acids is discussed.     

Deoxyribonucleic acid synthesis in isolated DNA-membrane complexes

The DNA synthesized by isolated Escherichia coli DNA-membrane complexes has been analysed by centrifugation techniques. The in vitro synthesized DNA sediments after deproteinization, in part with the parental bulk DNA and in part as a spectrum of fragments with sedimentation coefficients between 10 and 25 s. The fragments are, at least partially, precursors to the fast sedimenting DNA. The DNA fragments are mostly double stranded and contain (i) parental DNA pieces non-covalently bound to newly synthesized DNA strands of similar length, as well as (ii) one well-defined fraction in which parental DNA and newly synthesized DNA are covalently joined. The results are discussed in terms of the “prefork synthesis” model of Haskell & Davern (1969).     

Model membranes: Spherical shells bounded by one bimolecular layer of phospholipids

Summary The conditions are described which lead to the formation of small spherical vesicles bounded by singlelipid membranes. These membranes appear as triple-layered structures in electron micrographs and thus resemble theunit membrane configuration of cellular boundaries. The theory of light scattering by spherical shells is found applicable to the vesicles described here; size determinations by electron microscopy (r=195 Å) and by light scattering (r=225 Å) give reasonably close values. The refractive index of the membranes as determined by differential refractometry is found to be 1.46 for green incident light of wavelength 5461 Å.The results of this work were presented in part at the Symposium on Biophysical Aspects of Permeability, Jerusalem, Israel, July 2–9, 1968.I gratefully acknowledge the help provided me during this work by Dr Walter Stoeckenius, and I also thank Dr Dominic Dziewiatkowski for the permission to use his light scattering photometer.This work was supported by grant No. GB-4871 from the National Science Foundation.     

NMR in vitro measurements: a quality control study of the RADX table-top spectrometer

A series of experiments was performed to evaluate the accuracy and reproducibility of relaxation values obtained by two RADX table-top spectrometers operating at 5 MHz (R5) and 10 MHz (R10) respectively. The output (T1, T2, and proton content) of each machine was compared (for tissue specimens and paramagnetic solutions) to reference spectrometers. In the range of tissue T1's, R5 overestimates T1 by approx. 10% and R10 underestimates by approx. 23%. For tissue specimens, the T2 output of both machines is within 3% of the reference facility. Proton content values correlate well with the % wet weight of tissues (y = .46x + 24, r = .85) but accuracy deteriorates badly if tissue T1 greater than 400 msec or T2 greater than 300 msec. The output of both machines is accurate and reproducible within 5% over the range of tissue relaxation values (biological fluids excluded).