Gap-junctional coupling between neutrophils and endothelial cells: a novel modulator of transendothelial migration

Communication between leukocytes and endothelial cells is crucial for inflammatory reactions. Paracrine cross-talk and outside-in signaling (via adhesion molecules) have been characterized as communication pathways to date. As leukocytes and endothelial cells express connexins, we considered intercellular communication via gap junctions an intriguing additional concept. We found that gap-junctional coupling between neutrophils and endothelium occurred in a time-dependent, bidirectional manner and was facilitated by adhesion. After blockade of connexins, transmigration of neutrophils through the endothelial layer was enhanced, and the barrier function of cell monolayers was reduced during transmigration. Tumor necrosis factor alpha decreased coupling. In the presence of connexins, transmigration of neutrophils did not alter permeability. Thus, neutrophils couple to endothelium via gap junctions, functionally modulating transmigration and leakiness. Gap-junctional coupling may be a novel way of leukocyte-endothelial communication.     

Epidermal growth factor and trefoil factor family 2 synergistically trigger chemotaxis on BEAS-2B cells via different signaling cascades

Injured areas of the respiratory epithelium are subject to rapid repair by the migration of adjacent epithelial cells, a process termed "restitution". Rapid re-epithelialization is promoted by interactions between migrating cells and the extracellular matrix proteins. Furthermore, epidermal growth factor (EGF) as well as trefoil factor family (TFF) peptides are well known regulators of epithelial restitution due to their motogenic effects. Migration of the human bronchial epithelial cell line BEAS-2B in modified Boyden chambers was used as a model system for airway restitution. EGF or recombinant human TFF2 or TFF3 showed mainly chemotactic activity. The motogenic response was strictly dependent upon a haptotactic substrate, but to different degrees. EGF induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, c-Jun-N-terminal kinase, p38, Akt, and p70S6K in BEAS-2B cells. Using specific inhibitors, the signaling cascades responsible for the motogenic response were shown to differ drastically when EGF was compared with TFF2. The motogenic effect of TFF2 was previously demonstrated to depend on ERK1/2 and protein kinase C activation; whereas the EGF-triggered motogenic response was completely independent of ERK1/2 activation but sensitive to the inhibition of phosphoinositide 3-kinase, p38, protein kinase C, or nuclear factor kappaB. However, the motogenic effects of EGF and TFF2 are additive. These data suggest that luminal EGF and TFF peptides can act synergistically in the human respiratory epithelium to enhance rapid repair processes in the course of diseases such as asthma.     

Long-range DNase I hypersensitivity mapping reveals the imprinted Igf2r and Air promoters share cis-regulatory elements

Epigenetic mechanisms restrict the expression of imprinted genes to one parental allele in diploid cells. At the Igf2r/Air imprinted cluster on mouse chromosome 17, paternal-specific expression of the Air noncoding RNA has been shown to silence three genes in cis: Igf2r, Slc22a2, and Slc22a3. By an unbiased mapping of DNase I hypersensitive sites (DHS) in a 192-kb region flanking Igf2r and Air, we identified 21 DHS, of which nine mapped to evolutionarily conserved sequences. Based on the hypothesis that silencing effects of Air would be directed towards cis regulatory elements used to activate genes, DHS are potential key players in the control of imprinted expression. However, in this 192-kb region only the two DHS mapping to the Igf2r and Air promoters show parental specificity. The remaining 19 DHS were present on both parental alleles and, thus, have the potential to activate Igf2r on the maternal allele and Air on the paternal allele. The possibility that the Igf2r and Air promoters share the same cis-acting regulatory elements, albeit on opposite parental chromosomes, was supported by the similar expression profiles of Igf2r and Air in vivo. These results refine our understanding of the onset of imprinted silencing at this cluster and indicate the Air noncoding RNA may specifically target silencing to the Igf2r promoter.     

Phenotypic characterization of human chondrocyte cell line C-20/A4: a comparison between monolayer and alginate suspension culture

DNA microarray analysis was used to investigate the molecular phenotype of one of the first human chondrocyte cell lines, C-20/A4, derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen. Clontech Human Cancer Arrays 1.2 and quantitative PCR were used to examine gene expression profiles of C-20/A4 cells cultured in the presence of serum in monolayer and alginate beads. In monolayer cultures, genes involved in cell proliferation were strongly upregulated compared to those expressed by human adult articular chondrocytes in primary culture. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated after culture in alginate beads, consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. Also, nexin-1, vimentin, and IGFBP-3, which are known to be expressed by primary chondrocytes, were differentially expressed in our study. Consistent with the proliferative phenotype of this cell line, few genes involved in matrix synthesis and turnover were highly expressed in the presence of serum. These results indicate that immortalized chondrocyte cell lines, rather than substituting for primary chondrocytes, may serve as models for extending findings on chondrocyte function not achievable by the use of primary chondrocytes.     

Distinct sites of action of clostridial neurotoxins revealed by double-poisoning of mouse motor nerve terminals

(1) We investigated the effects of single- and double-poisoning with tetanus toxin (TeTx), botulinum neurotoxin type A (BoTx A) and botulinum neurotoxin type B (BoTx B) on spontaneous and nerve-evoked quantal transmitter release at motor endplates of the triangularis sterni preparation of the mouse. (2) Inhibitory effects of TeTx and BoTx B on spontaneous and nerve-evoked transmitter release were very similar, except that the action of BoTx B required 500-fold lower concentrations and was less dependent on temperature. BoTx A caused stronger inhibition of quantal release than TeTx or BoTx B, but was comparatively much easier counteracted by 4-aminopyridine (4-AP). (3) In contrast to BoTx A, with TeTx or BoTx B the increase of transmitter release following onset of 50 Hz nerve stimulation was delayed for a few seconds and synaptic latencies of quanta showed large variations. This release pattern was also evident in all double-poisoning experiments, regardless of intoxication sequence. (4) Inhibition of evoked release was found to be slightly stronger with TeTx than with BoTx B, so the amount of nerve-evoked quanta released after double-poisoning with any sequence of these toxins always approached that of TeTx. In no case supraadditive actions were observed. (5) A strong reduction of evoked quanta was observed when BoTx A was applied in addition to either of the two other toxins. With reversed poisoning sequences (BoTx A-TeTx or BoTx A-BoTx B) the resulting values remained at the extremely low level of BoTx A. (6) In the presence of 4-AP double-poisoning with any combination between BoTx A and TeTx or BoTx B (regardless of intoxication sequence) revealed supra-additive effects, since the number of quanta released was considerably lower than that obtained with any of the toxins alone (in the presence of 4-AP). (7) Our results indicate that tetanus toxin and botulinum toxin type B have a common site of action which is different and independent from that of botulinum toxin type A.This is part of the thesis of M. G. to be presented to the Fachbereich Humanmedizin, Justus-Liebig-Universität Gießen     

Pre-screening via sleep logs in sleep-disordered patients—Adaptational effects, yes or no?

Summary Question of the study  Sleep logs are common tools in sleep research and clinical routine. Usually sleep logs have to be completed during a 2-week period, with the first week serving as an adaptation to the instrument itself. In the present study, we investigated whether there is indeed such an adaptation bias or not. Patients and methods  A total of 236 chronically sleep-disordered outpatients completed the standardized sleep log ‘Abend-Morgen-Protokoll’ during a 2-week pre-screening period prior to the first visit in our sleep ambulance. Two sets of items were established, the ‘instrumental’ and the ‘therapeutic’ set. The respective ratings of the first and second week (week A, B) were compared to evaluate clinically relevant changes. Results  The ratings of several ‘instrumental’ items significantly differed between week A and B. However, these changes—on average—were only marginal and therefore of little clinical importance. Regarding the ‘therapeutic’ set of items, no systematic variations could be ascertained over the assessment period. Conclusion  The present investigation could not confirm the presence of adaptation biases (instrumental, therapeutic) in a large sample of chronically sleep-disordered outpatients. Therefore, we consider a 1-week pre-screening period via sleep log as sufficient for the diagnostic process in these patients.      

A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA

To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that converted the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed.