Group B streptococcus (GBS) is surrounded by a capsule. However, little is known about peptidoglycan metabolism in these bacteria. In the present study, a 65 kDa protein was isolated from the culture supernatant of GBS and N-terminally sequenced, permitting isolation of the corresponding gene, termed bsp. The bsp gene was located close to another gene, designated femH, and reverse transcription-PCR revealed a bicistronic transcriptional organization for both genes. The Bsp protein was detected in the culture supernatant from 31 tested clinical isolates of GBS, suggesting a wide distribution of Bsp in these bacteria. Overexpression of bsp resulted in lensshaped GBS cells, indicating a role for bsp in controlling cell morphology. Insertional disruption of femH resulted in a reduction of the L-alanine content of the peptidoglycan, suggesting that femH is involved in the incorporation of L-alanine residues in the interpeptide chain of the peptidoglycan of GBS.
The EphB/ephrinB receptor-ligand system is pivotal for the development of the embryonic vasculature and for angiogenesis in the adult organism. We observed that (i) the expression of ephrinB2 and ephrinB1 is up-regulated in capillaries during inflammation, that (ii) these ligands are localised on the luminal endothelial surface, and that (iii) they interact with the ephrinB-receptor EphB2 on monocyte/macrophages. This study delineates the impact of ephrinB-mediated reverse signalling on the integrity and proinflammatory differentiation of the endothelium. To this end, in vitro analyses with human cultured endothelial cells reveal that knockdown of ephrinB2 or ephrinB1 impairs monocyte transmigration through the endothelium. While ephrinB2 but not ephrinB1 interacts with PECAM-1 (CD31) in this context, reverse signalling by ephrinB1 but not ephrinB2 elicits a c-Jun N-terminal kinase (JNK)-dependent up-regulation of E-selectin expression. Furthermore, treatment of endothelial cells with soluble EphB2 receptor bodies or EphB2-overexpressing mouse myeloma cells links ephrinB2 to PECAM-1 and induces its Src-dependent phosphorylation while diminishing Src homology phosphotyrosyl phosphatase-2 (SHP-2) activity and increasing endothelial cell permeability. We conclude that extravasation of EphB2 positive leukocyte populations is facilitated by lowering the integrity of endothelial cell junctions and enhancing the pro-inflammatory phenotype of the endothelium through activation of ephrinB ligands.
The migration of oligodendrocyte progenitor cells (OPCs) to the white matter is an indispensable requirement for an intact brain function. The mechanism of cell migration in general is not yet completely understood. Nevertheless, evidence is accumulating that besides the coordinated rearrangement of the cytoskeleton, a finetuned interplay of ion and water fluxes across the cell membrane is essential for cell migration. One part of a general hypothesis is that a local volume increase towards the direction of movement triggers a mechano-activated calcium influx that regulates various procedures at the rear end of a migrating cell. Here, we investigated cell volume changes of migrating OPCs using scanning ion conductance microscopy. We found that during accelerated migration OPCs undergo an increase in the frontal cell body volume. These findings are supplemented with time lapse calcium imaging data that hint an increase in calcium content the frontal part of the cell soma.
BackgroundCell volume determination plays a pivotal role in the investigation of the biophysical mechanisms underlying various cellular processes. Whereas light microscopy in principle enables one to obtain three dimensional data, the reconstruction of cell volume from z-stacks is a time consuming procedure. Thus, three dimensional topographic representations of cells are easier to obtain by scanning probe microscopical measurements.ResultsWe present a method of separating the cell soma volume of bipolar cells in adherent cell cultures from the contributions of the cell processes from data obtained by scanning ion conductance microscopy. Soma volume changes between successive scans obtained from the same cell can then be computed even if the cell is changing its position within the observed area. We demonstrate that the estimation of the cell volume on the basis of the width and the length of a cell may lead to erroneous determination of cell volume changes.ConclusionsWe provide a new algorithm to repeatedly determine single cell soma volume and thus to quantify cell volume changes during cell movements occuring over a time range of hours.
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