PubMed Health⌕ Search

Biomedical subjects

P Clissold

Publications and source records attributed to P Clissold.

9 recordsLinked to original sources
PubMed1997

Expression of beta-catenin and the adenomatous polyposis coli tumour suppressor protein in mouse neocortical cells in vitro.

Beta-catenin is known to associate with the tumour suppressor protein adenomatous polyposis coli (APC), which is highly expressed in developing brain. We have therefore investigated the distribution of beta-catenin and APC in primary cultures of mouse neocortex. Western blotting demonstrated the presence of a single beta-catenin species in our cultures. Immunocytochemistry showed that beta-catenin was plasma membrane associated and concentrated in growth cones in cultured neurons. The APC tumour suppressor protein was also concentrated in growth cones. In glial cells, beta-catenin was localised at cell-cell contacts in a manner similar to that previously described in other cell types. This data suggests a role for both APC and beta-catenin in neuronal growth cones, and for beta-catenin in the formation of cell to cell contacts between glia.

Adenomatous Polyposis Coli↗
PubMed1997

The cellular distribution of the adenomatous polyposis coli tumour suppressor protein in neuroblastoma cells is regulated by microtubule dynamics.

The adenomatous polyposis coli tumour suppressor protein is highly expressed in developing rodent brain, but its function is unclear. Recent studies have suggested a role for this protein in regulating microtubule dynamics. Neuro 2A mouse neuroblastoma cells were previously thought not to express this protein. Using immunochemical techniques, this report corrects this observation. Immunoreactive bands of a size consistent with that of the full-length protein were observed by western blotting. Using immunocytochemistry, punctate immunoreactivity localized to areas of the cell containing microtubules, particularly neurite growth cones, in a distribution suggesting a role in neuritogenesis and growth cone extension. The protein did not localize to actin-rich cellular structures, and perturbation of the actin cytoskeleton had no effect upon this distribution. Treatment of cells with taxol to stabilize microtubules caused the concentration of the immunoreactive puncta to the tips of microtubules and areas along the axis of potential microtubule assembly. Treatment of cells with the microtubule disrupting reagent nocodazole showed that over shorter times the punctate distribution was not dependent upon polymerized microtubules. However, at longer incubation times a decrease in punctate immunostaining was observed. These results indicate that the intracellular distribution of the adenomatous polyposis coli protein is dependent upon microtubule but not actin dynamics. A role for this protein in the regulation of directed microtubule assembly is suggested.

Adenomatous Polyposis Coli↗
PubMed1994

Herpes simplex virus 1 (HSV-1) helper co-infection affects the distribution of an amplicon encoded protein in glia.

HSV-1 derived amplicons expressing cytoplasmic beta-galactosidase (pA-SF1), or plasma membrane targeted TIMP-Thy1 (pA-TT1), were used to transduce glial cells in vitro. By monitoring the expression of reporter genes from both amplicons and helper virus, we determined that many cells were infected by both particles. In glial cells infected only by pA-SF1 beta-galactosidase immunoreactivity was restricted to the cytoplasm; co-infection with helper HSV-1 (wild type), resulted in additional nuclear beta-galactosidase immunoreactivity. Co-infection of cells with amplicon pA-TT1 and helper virus did not affect the plasma membrane localization of TIMP/Thy1. Thus, co-infection with wild type helper virus altered the localization of an amplicon encoded cytoplasmic, but not plasma membrane protein.

Cell Membrane↗
PubMed1994

Use of recombinant vectors derived from herpes simplex virus 1 mutant tsK for short-term expression of transgenes encoding cytoplasmic and membrane anchored proteins in postmitotic polarized cortical neurons and glial cells in vitro.

We constructed three recombinant vectors derived from the herpes simplex virus type 1 mutant tsK, each of which contained a different transgene under the control of the herpes simplex virus type 1 immediate early 3 promoter inserted into the thymidine kinase locus: the prokaryotic enzymes beta-galactosidase and chloramphenicol acetyl transferase, and a fusion gene consisting of human tissue inhibitor of metalloproteinases linked to the last exon of Thy-1, which encodes for a glycosyl-phosphatidyl-inositol membrane anchor. Infection of postmitotic neocortical and hippocampal neurons in low-density primary cultures with these vectors, achieved reliable expression of all three foreign gene products in various neocortical cell types, e.g. pyramidal neurons, non-pyramidal neurons, and glial cells. The percentage of neurons expressing transgenes ranged from 1 to 46% depending on the multiplicity of infection (highest assayed = 5); the percentage of glial cells expressing transgenes ranged from 0.5 to 98% (highest multiplicity assayed = 3.4). Expression of transgenes could be detected for up to three days in approximately 20% of neurons infected at a multiplicity of infection of 1. Infection of neurons with tk K-derived recombinant vectors inhibited their protein synthesis by 40-50% at a multiplicity of infection of 10, but no effect was observed at a multiplicity of infection of 1. Infection of glial cells with the same vectors at a multiplicity of infection of 1 inhibited protein synthesis by more than 90%. Analysis of neuronal viability at different times post-infection indicated that more than 98% of neurons expressing transgenes 48 h post-infection were viable. Thus, low-density neuronal cultures can be used to assess the efficiency of herpes simplex virus type 1-derived gene transfer vectors and transgene expression in developing cortical postmitotic cells, before and after they establish polarity. In addition, we show that two cytoplasmic enzymes, beta-galactosidase and chloramphenicol acetyl transferase, are able to diffuse freely in the cytoplasm reaching even growth cones in young neurons, while the chimeric protein tissue inhibitor of metalloproteinases/Thy-1 is correctly targeted to the plasma membrane via a glycosyl-phosphatidylinositol anchor. This model system should be useful for investigation of cellular and molecular aspects of the development and establishment of neuronal polarity, as well as for analysis of signals involved in protein targeting in postmitotic neurons.

Amino Acid Sequence↗
PubMed1994

HSV1 vectors to study protein targeting in neurones: are glycosyl-phosphatidylinositol anchors polarized targeting signals in neurones?

In order to characterize protein targeting signals in polarized postmitotic cortical neurones in vitro, we have developed recombinant and amplicon type vectors derived from herpes simplex virus 1 (HSV1) to transfer genes into these cells. We examined the targeting of both bacterial proteins, which lack specific targeting signals, as well as recombinant proteins containing mammalian targeting sequences, i.e. the sequence encoding for the addition of a glycosyl-phosphatidyl inositol (GPI) membrane anchor. Utilizing both HSV1 recombinant and amplicon vectors we demonstrate that while a GPI anchor is able to direct a foreign protein to the plasma membrane, the recombinant protein is targeted mainly to the dendritic, but also to the axonal plasma membrane; i.e. its distribution is not completely polarized. Thus, GPI anchors do not function as dominant polarized targeting signals in neurones, as they do in polarized epithelial cell lines.

Cells, Cultured↗
PubMed1988

Chemical synthesis, cloning and expression in mammalian cells of a gene coding for human tissue-type plasminogen activator.

A 1610-bp DNA duplex coding for human tissue-type plasminogen activator has been chemically synthesized using the phosphoramidite procedure, adapted for a custom-built gene synthesizer. The synthesizer, which was designed for both simplicity and speed, permits the rapid construction of relatively large genes and compares favorably in speed with alternative cDNA isolation procedures. The plasminogen activator gene has been expressed in mammalian cells and shown to produce authentic protein by an immuno-activity assay.

Base Sequence↗