Biomedical subjects

S E Webb

Publications and source records attributed to S E Webb.

23 records · Page 2Linked to original sources

PubMed1996

Contractile protein system in the asexual stages of the malaria parasite Plasmodium falciparum.

F-actin was detected in asexual-stage Plasmodium falciparum parasites by fluorescence microscopy of blood films stained with fluorescent phalloidin derivatives. F-actin was present at all stages of development and appeared diffusely distributed in trophic parasites, but merozoites stained strongly at the poles and peripheries. No filament bundles could be discerned. A similar distribution was obtained by immunofluorescence with 2 polyclonal anti-actin antibodies, one of which was directed against a peptide sequence present only in parasite actin (as inferred from the DNA sequence of the gene). A monoclonal anti-actin antibody stained very mature or rupturing schizonts but not immature parasites. Myosin was identified in immunoblots of parasite protein extracts by several monoclonal anti-skeletal muscle myosin antibodies, as well as by a polyclonal antiserum directed against a consensus conserved myosin sequence (IQ motif). The identity of the polypeptides recognised by these antibodies was confirmed by overlaying blots with biotinylated F-actin. The antiserum and one of the monoclonal antibodies were used in immunofluorescence studies and were found to stain all blood-stage parasites, with maximal intensity towards the poles of merozoites. Our results are consistent with the presence of an actomyosin motor system in the blood-stage malaria parasite.

Actins↗

PubMed1996

Direct observation and quantification of macrophage chemoattraction to the growth factor CSF-1.

The cloned mouse macrophage cell line, BAC1.25F, resembles primary macrophages in its dependence on colony stimulating factor-1 (CSF-1) for both viability and proliferation. Re-addition of CSF-1 to cytokine-deprived cells, which are rounded with diffusely organised F-actin, stimulates rapid cell spreading and cell polarisation. Using the Dunn chemotaxis chamber the movement of stimulated macrophages was monitored over a 2 hour period. Cells restimulated with 1.32 nM human recombinant CSF-1 migrated at a mean rate of 7.71 microns per hour, but showed no directional preferences. In a linear concentration gradient of CSF-1, cytokine-deprived cells were again stimulated to migrate and the mean rate of cell motility, at 6.88 microns per hour, was not significantly different from that measured in an isotropic environment of CSF-1. However, there was a strong preference for the cells to orientate so that their long axes aligned with the CSF-1 gradient and they migrated preferentially towards the source of CSF-1. Migrating cells contained abundant F-actin within the leading lamellae as judged by confocal imaging of fluorescent phalloidin, but the actin was not arranged into stress fibre-like structures. These data support the proposition that CSF-1 is both a chemokinetic and chemotactic agent for macrophages. Tumour necrosis factor (TNF-alpha) failed to stimulate cell migration and thus was neither chemokinetic nor a chemotactic agent. However, cells exposed to a dual concentration gradient of both TNF-alpha and CSF-1 did migrate successfully, although the chemotactic response to CSF-1 was abolished.

Actins↗

PubMed1996

Differential expression of the soluble 170 kDa brain protein in the fetal and adult human brain.

The expression of the soluble 170 kDa brain protein (BP170) was studied in the developing human brain. Using immunohistochemical methods, it was possible to demonstrate that BP170 was expressed very strongly in the visual cortex up to approximately 27 weeks of gestation. After this time, the level of expression was reduced so that by adulthood the levels of BP170 were negligible. Similarly, BP170 was expressed in the hippocampus at all stages of development; however, unlike in the visual cortex, this protein was still apparent, albeit at low levels, in the adult. In addition, BP170 was co-expressed with tyrosine hydroxylase which suggests its possible role in the dopamine pathway.

Adult↗

PubMed1995

Migration of myogenic cells from the somites to the fore-limb buds of developing mouse embryos.

In this study, we have isolated newly formed somites from the caudal regions of 8.5 day mouse embryos and transplanted them orthotopically into correspondingly staged hosts at the level of the prospective limb-forming region. The experimental embryos were then cultured intact for 32-36 hr. The donor somites used were pre-labelled with DiI, a fluorescent lipophilic dye, or were obtained from transgenic embryos that carried a 1 kb 5' regulatory sequence of the desmin gene linked to the gene encoding Escherichia coli beta-galactosidase. The transgene is specifically expressed in skeletal muscles (Li et al. [1993] Development 117:947-959). The aim of these experiments was to show definitively that the musculature of the mammalian limb is derived from the somites. The results demonstrated that DiI-labelled cells from the implanted somites were able to invade the proximal region of the fore-limb bud during the course of development. The use of transgenic somites as grafts confirmed that some of the somitic cells found in the limbs were myogenic cells. To determine whether the displacement of somitic cells is an active or passive process, somatopleure obtained from the prospective limb-forming regions of day 8.5 day embryos was implanted into 8.5 day hosts. We did not detect the presence of DiI-labelled somatopleural cells in the fore-limb after 32-36 hr of culture. This suggests that somitic cells reached the limb bud via active locomotion rather than as a result of being passively dragged there, as the limb elongates during development. In addition, we injected latex beads into the somites, as probes, to determine whether extracellular matrix-driven translocation plays a role in driving the somitic cells to the limb bud. In a majority of the specimens examined, we could not detect the presence of these beads in the limb bud. However, in the trunk of these embryos, the beads were found dispersed throughout the ventral neural crest pathway.

Animals↗

PubMed1995

Desmin transgene expression in mouse somites requires the presence of the neural tube.

Transgenic mice were used to study the effect of the neural tube on somite myogenesis. These mice express a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene which codes for E. coli beta-galactosidase. In order to determine whether the developmental fate of cells, specifically the prospective myogenic population, in newly developed somites was pre-determined, newly formed somites were isolated from the caudal region of day 9.5 transgenic embryos and transplanted into 8.5 day non-transgenic host embryos. Even though the implanted somites were not oriented in the host embryos, all the specimens examined developed normally at the graft site forming a dermatome, myotome and sclerotome in the correct anatomical positions. The myotome even expressed the desmin transgene. In addition, we isolated the 3 most caudal somites, that is, the most recently developed somites, from day 9.5 transgenic embryos and maintained them on gelatin-coated coverslips in culture for up to 4 days. While these somite explants did not develop myoblasts, it was possible to induce myogenesis by introducing pieces of neural tube into the explant cultures. These results suggest that the developmental fate of cells within the newly formed somite is not predetermined, but is dependent on the microenvironment surrounding the developing somite.

Animals↗