N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.     

Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes

A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise regional chromosomal origin of the markers. Following this approach, we tested 2–14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY, +mar₁,+mar₂] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].     

Peptide Bβ15-42 Preserves Endothelial Barrier Function in Shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn^−/− mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.     

The regeneration of neuromuscular junctions during spontaneous re-innervation of the rat diaphragm

Summary Electron microscopic observations have been made on the regeneration of neuromuscular junctions during spontaneous re-innervation of the rat diaphragm, following unilateral transsection of the phrenic nerve. 3 and 4 weeks after denervation motor end plates displayed the pattern of almost complete degeneration, i.e. persisting subneural foldings, deprived of neural contact and covered with collagen fibrils and fibrocytes. From observations at 5, 10 and 24 weeks after denervation the following sequence of events could be established: a few small axon terminals, accompanied by Schwann cells, became apposed to subneural folds, while most foldings were covered initially by Schwann cells or still by collagen fibrils. Gradually an increasing number of subneural folds came into contact with axon terminals. At 24 weeks all junctions displayed the pattern of a mature motor end plate. In the majority of regenerating neuromuscular junctions single dense-cored vesicles of approximately 900–1200 Å were present in axon terminals.It is concluded that under the present conditions restoration of neuromuscular transmission is accomplished by a re-innervation of the preserved subneural apparatuses of former junctions by regenerating axons. The significance of the occurrence of dense-cored vesicles in regenerating motor end plates is discussed.This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.     

On the ultrastructure of the rat anococcygeus muscle

Cell and Tissue Research - The rat anococcygeus muscle, which is known from previous functional and histochemical investigations to be a smooth muscle with a dense adrenergic innervation, was...     

A new method to measure nitrate/nitrite with a NO-sensitive electrode

There are different methods to measure the unstable moleculenitric oxide (NO). We will describe a new sensitive method to measure NO by reconversion of nitrate/nitrite to NO, which will bedetermined with an amperometric Clark-type electrode. Nitrate andnitrite are the degradation products of NO. First, nitrate isenzymatically converted to nitrite with the use of the nitrate reductase. Second, nitrite is reduced to equimolar NO concentrations byan acidic iodide solution. The detection limit of the electrode in anaqueous solution was 2 nmol/l NO (meaning the threshold was dependingon the volume added: 500 µl of a 0.2 µmol/l nitrite solution addedto a 10-ml bath). This method provides the ability to assess basal andagonist-stimulated NO releases of different biological models. Wemeasured basal and carbachol-stimulated NO release of nativeendothelial cells from porcine coronary arteries and porcine aorticendothelial cell cultures. Moreover, it was possible to measure thenitrate/nitrite concentration in the coronary effluent of a guinea pigheart. In conclusion, we present a valid, highly sensitive new methodof measuring nitrite/NO in biological systems with a commerciallyavailable electrode.

     

Aufenthaltsdauern explorierender Meisen: Habitat- und Artspezifit?t

Ohne Zusammenfassung

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Patch residence times of exploring tits: habitat and species specificity

     

Frequent diploidisation of haploid Armillaria ostoyae strains in an outdoor inoculation experiment

Very little is known about the biology and ecology of haploid Armillaria strains in nature. In this outdoor inoculation experiment, we assessed the virulence of six haploid Armillariaostoyae strains along with their diploid parent towards 2-year-old seedlings and 4-year-old saplings of Norway spruce (Picea abies), and determined their ability to colonise freshly cut stumps. As inoculum source an Armillaria-colonised hazelnut (Corylus avellana) stem segment was inserted into the soil substrate. Re-isolations from mycelial fans at the root collar of infected trees or stumps were made. Surprisingly, not a single haploid re-isolate could be recovered. Microsatellite genotyping of 133 re-isolates suggests that the inoculated haploid strains were diploidised either by mating propagules (basidiospores or haploid mycelia) already present in the soil substrate or naturally disseminated in the course of the experiment from nearby forests. Consequently, no conclusion about the infectious ability of haploid Armillaria mycelia under natural conditions can be drawn. Nonetheless, the diploid half-sib families resulting from the diploidisation showed varying degrees of virulence, with a high correlation between the experiment with 2-year-old seedlings and 4-year-old saplings. Despite extensive genotyping of re-isolates, no evidence for somatic recombination between haploid mating propagules and diploidised mycelia was detected, suggesting that this is an uncommon phenomenon in A. ostoyae.