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Biomedical subjects

P Doherty

Publications and source records attributed to P Doherty.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mapping regions of the beta1 integrin cytoplasmic domain involved in migration and survival in primary oligodendrocyte precursors using cell-permeable homeopeptides.

The mapping of regions within integrin cytoplasmic domains responsible for the different effects on cell behaviour is an important part of an analysis of integrin-mediated signalling. In order to facilitate this analysis in primary cells, we have used cell-permeable homeopeptides to deliver sequences mimicking parts of the integrin beta1 cytoplasmic domain into the cell. In a study using oligodendrocyte precursors, the cells that give rise to myelin-forming oligodendrocytes during CNS development, we show that these peptides can be used to manipulate beta1 integrin signalling and that the regions of the cytoplasmic domain involved in migration and survival are distinct. Peptides mimicking the N-terminal portion of the cytoplasmic domain previously implicated in binding to Focal Adhesion Kinase (FAK) induce apoptosis, while peptides mimicking more C-terminal sequences do not cause cell death. In contrast they show that the NPIY sequence, the N-terminal one of two NPXY motifs previously implicated in signalling, is involved in migration. Peptides containing this sequence promote migration while alteration of NPIY to NPIA makes the peptide inhibitory to migration. Our results show that these peptides represent a novel approach to integrin signalling that allow rapid definition of critical cytoplasmic sequences in primary cells.

Amino Acid Sequence↗
PubMed1999

Evidence for and against a pivotal role of PI 3-kinase in a neuronal cell survival pathway.

PI 3-kinase has emerged as a key enzyme for regulating neuronal cell survival. However, it has not as yet been demonstrated whether activation of the endogenous pool of the enzyme, that is regulated by the p85 subunit, is sufficient to promote a survival response. It is also not known whether the FGF family of growth factors promote survival via a PI 3-kinase-dependent pathway. We have previously developed a cell permeable p85 binding peptide and shown that it can stimulate a mitogenic response in muscle cells that is dependent on a PI 3-kinase/p70 S6 kinase pathway. In the present study we show that this peptide can rescue cerebellar granule cells from death induced by serum deprivation and that this response is comparable to a growth factor response (FGF2). Experiments with wortmannin, LY294002, and rapamycin suggest that the peptide survival response is dependent on PI 3-kinase activity, but not p70 S6 kinase activity. The peptide response was correlated with a PI 3-kinase-dependent phosphorylation of Akt, an established downstream effector in the PI 3-kinase survival cascade. In contrast to the survival response stimulated by the p85 binding peptide, the response stimulated by FGF2 was not inhibited by wortmannin or LY294002, nor was it associated with phosphorylation of Akt. Thus we can conclude that activation of the endogenous pool of PI 3-kinase that is regulated by p85 is sufficient for cell survival; however, growth factors such as FGF2 can clearly support survival in a PI 3-kinase-independent manner.

Animals↗
PubMed1999

A soluble version of the receptor-like protein tyrosine phosphatase kappa stimulates neurite outgrowth via a Grb2/MEK1-dependent signaling cascade.

Receptor-like protein tyrosine phosphatase kappa (RPTPkappa) is expressed in the nervous system in a manner consistent with a role in axonal growth and guidance. The extracellular domain of RPTPkappa shares structural features with cell adhesion molecules and can support homophilic adhesion. In the present study we produced a soluble Fc-chimeric protein containing the full extracellular domain of RPTPkappa. Following affinity capture, the RPTPkappa-Fc was shown to promote the aggregation of Covasphere beads, confirming its homophilic binding activity. When added to cultures of cerebellar neurons as a soluble molecule, the RPTPkappa chimera stimulated neurite outgrowth. The neurite outgrowth response was substantially inhibited by a cell-permeable peptide inhibitor of Grb2 and by PD 098059, a drug that has been used to inhibit MEK1 activation in a wide range of cell types. These results demonstrate that RPTPkappa can stimulate neurite outgrowth and provide evidence that this might involve the coupling of Grb2 to a MAPK signal transduction cascade.

3T3 Cells↗
PubMed1999

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.

Amino Acid Sequence↗
PubMed1999

Use of antipsychosis and adjunctive medications by an inner urban community psychiatric service.

OBJECTIVE: The aim of this paper is to survey patterns of use of new generation and conventional antipsychosis and adjunctive drugs by an inner urban community psychiatric service. METHOD: All prescriptions for antipsychosis medications and all patients receiving these drugs in May 1998 were identified. Case record review yielded demographic and diagnostic data. Information was also obtained directly from prescribers. RESULTS: Of 859 patients, 77% received antipsychosis medication; 53% of prescriptions for antipsychotics were for new generation drugs: risperidone (42%), olanzapine (37%) and clozapine (21%). Mean doses were 4.1+/-2.5 mg (risperidone), 14.7+/-8.2 mg (olanzapine) and 377.4+/-178.9 mg (clozapine). Doses for men tended to be higher than those for women, but the differences were not significant. DSM-IV diagnosis was schizophrenia for 74% of patients on atypicals, but patients with other diagnoses were also being treated with these drugs. Risperidone was more commonly used in combination with benzodiazepines and anticholinergics than olanzapine and clozapine, while clozapine was less likely to be combined with antidepressants and mood stabilisers. Of the conventionals, 66% were in depot form, mostly because of non-compliance. Combinations of antipsychotics were prescribed to 13% of patients. CONCLUSION: New generation antipsychosis medications were prescribed more commonly than conventional drugs in this service for a wide range of diagnoses. Adjunctive medications were commonly utilised. These findings underline the clinical complexity of antipsychotic treatment in a changing environment.

Adult↗
PubMed1999

CAMs and FGF cause a local submembrane calcium signal promoting axon outgrowth without a rise in bulk calcium concentration.

Binding of basic fibroblast growth factor (bFGF) and cell adhesion molecules to the nerve cell membrane promotes axon outgrowth. This response can be blocked by antagonists of voltage-gated calcium channels, yet no change of cytosolic calcium concentration in the growth cone can be detected upon binding of the growth factor bFGF or the cell adhesion molecule L1. Using barium as a charge carrier, we show that bFGF and L1 open a calcium influx pathway in growth cones of rat sensory neurons without changing the membrane voltage. L1 does not activate influx in cells expressing a dominant negative mutant of the fibroblast growth factor receptor (FGFR) tyrosine kinase. FGFR-activated influx is blocked by specific antagonists of L- and N-type voltage-gated calcium channels and by an inhibitor of diacylglycerol lipase. We propose that both L1 and bFGF act via the FGFR to generate polyunsaturated fatty acids which in turn cause calcium channels to flicker open and shut. Short-lived domains of raised calcium at the cytosolic mouth of open channels activate axon outgrowth without raising bulk cytosolic calcium concentration. In confirmation of this model, the rapidly-acting calcium buffer BAPTA is significantly more effective at blocking FGF-induced axon outgrowth when compared with the slower buffer EGTA. Generation of short-lived calcium domains may provide a crucial mechanism for axon guidance during development and for promoting regeneration of damaged axons.

Animals↗
PubMed1999

Formation of polyglutamine inclusions in non-CNS tissue.

Huntington's disease (HD) is one of a class of inherited progressive neurodegenerative disorders that are caused by a CAG/polyglutamine repeat expansion. We have previously generated mice that are transgenic for exon 1 of the HD gene carrying highly expanded CAG repeats which develop a progressive movement disorder and weight loss with similarities to HD. Neuronal inclusions composed of the exon 1 protein and ubiquitin are present in specific brain regions prior to onset of the phenotype, which in turn occurs long before specific neurodegeneration can be detected. In this report we have extended the search for polyglutamine inclusions to non-neuronal tissues. Outside the central nervous system (CNS), inclusions were identified in a variety of post-mitotic cells. This is consistent with a concentration-dependent nucleation and aggregation model of inclusion formation and indicates that brain-specific factors are not necessary for this process. To possibly gain insights into the wasting that is observed in the human disease, we have conducted a detailed analysis of the timing and progression of inclusion formation in skeletal muscle and an investigation into the cause of the severe muscle atrophy that occurs in the mouse model. The formation of inclusions in non-CNS tissues will be particularly useful with respect to in vivo monitoring of pharmaceutical agents selected for their ability to prevent polyglutamine aggregation in vitro, without the requirement that the agent can cross the blood-brain barrier in the first instance.

Animals↗
PubMed1999

Evidence for collapsin-1 functioning in the control of neural crest migration in both trunk and hindbrain regions.

Collapsin-1 belongs to the Semaphorin family of molecules, several members of which have been implicated in the co-ordination of axon growth and guidance. Collapsin-1 can function as a selective chemorepellent for sensory neurons, however, its early expression within the somites and the cranial neural tube (Shepherd, I., Luo, Y. , Raper, J. A. and Chang, S. (1996) Dev. Biol. 173, 185-199) suggest that it might contribute to the control of additional developmental processes in the chick. We now report a detailed study on the expression of collapsin-1 as well as on the distribution of collapsin-1-binding sites in regions where neural crest cell migration occurs. collapsin-1 expression is detected in regions bordering neural crest migration pathways in both the trunk and hindbrain regions and a receptor for collapsin-1, neuropilin-1, is expressed by migrating crest cells derived from both regions. When added to crest cells in vitro, a collapsin-1-Fc chimeric protein induces morphological changes similar to those seen in neuronal growth cones. In order to test the function of collapsin-1 on the migration of neural crest cells, an in vitro assay was used in which collapsin-1-Fc was immobilised in alternating stripes consisting of collapsin-Fc/fibronectin versus fibronectin alone. Explanted neural crest cells derived from both trunk and hindbrain regions avoided the collapsin-Fc-containing substratum. These results suggest that collapsin-1 signalling can contribute to the patterning of neural crest cell migration in the developing chick.

Animals↗
PubMed1999

Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain.

Experiments involving tissue recombinations have implicated both early vertical and later planar signals in the specification and polarisation of the midbrain. Here we investigate the role of fibroblast growth factors in regulating these processes in the avian embryo. We show that Fgf4 is expressed in the notochord anterior to Hensen's node before transcripts for the earliest molecular marker of midbrain tissue in the avian embryo, En1, are detected. The presence of notochord is required for the expression of En1 in neural plate explants in vitro and FGF4 mimics this effect of notochord tissue. Subsequently, a second member of the fibroblast growth factor family, Fgf8, is expressed in the isthmus in a manner consistent with it providing a polarising signal for the developing midbrain. Using a retroviral vector to express En1 ectopically, we show that En1 can induce Fgf8 expression in midbrain and posterior diencephalon. Results of the introduction of FGF8 protein into the anterior midbrain or posterior diencephalon are consistent with it being at least part of the isthmic activity which can repolarise the former tissue and respecify the latter to a midbrain fate. However, the ability of FGF8 to induce expression of genes which have earlier onsets of expression than Fgf8 itself, namely En1 and Pax2, strongly suggests that the normal function of FGF8 is in maintaining patterns of gene expression in posterior midbrain. Finally, we provide evidence that FGF8 also provides mitogenic stimulation during avian midbrain development.

Animals↗
PubMed1998

Neurite outgrowth stimulated by neural cell adhesion molecules requires growth-associated protein-43 (GAP-43) function and is associated with GAP-43 phosphorylation in growth cones.

The mechanisms whereby cell adhesion molecules (CAMs) promote axonal growth and synaptic plasticity are poorly understood. Here we show that the neurite outgrowth stimulated by NCAM-mediated fibroblast growth factor (FGF) receptor activation in cerebellar granule cells is associated with increased GAP-43 phosphorylation on serine-41. In contrast, neither NCAM nor FGF was able to stimulate neurite outgrowth in similar neurons from mice in which the GAP-43 gene had been deleted by homologous recombination. Integrin-mediated neurite outgrowth was unaffected by GAP-43 deletion. Both neurite outgrowth and rapid phosphorylation of GAP-43 in isolated growth cones required the first three Ig domains of a NCAM-Fc chimera and were stimulated maximally at 5 micrograms/ml (approximately 50 nM). Likewise, GAP-43 phosphorylation in isolated growth cones also was stimulated by an L1-Fc chimera. Both neurite outgrowth and NCAM-stimulated GAP-43 phosphorylation were inhibited by antibodies to the FGF receptor and a diacylglycerol lipase inhibitor (RHC80267) that blocks the production of arachidonic acid in response to activation of the FGF receptor. Direct activation of the FGF receptor and the arachidonic acid cascade with either basic FGF or melittin also resulted in increased GAP-43 phosphorylation. These data suggest that the stimulation of neurite outgrowth by NCAM requires GAP-43 function and that GAP-43 phosphorylation in isolated growth cones occurs via an FGF receptor-dependent increase in arachidonic acid.

3T3 Cells↗
PubMed1998

Identification of a signaling pathway activated specifically in the somatodendritic compartment by a heparan sulfate that regulates dendrite growth.

In two earlier reports we demonstrated that natural heparan sulfate, but not dermatan or chondroitin sulfate glycosaminoglycans, stimulate axonal elongation and inhibit dendrite growth in vitro (Lafont et al., 1992). The latter specific effect on dendrite elongation was reproduced by chemically synthesized heparan sulfates and by SR 80037A, a purified sulfated and hexanoylated heparin fragment (Lafont et al., 1994). Adding radioactive SR 80037A to purified neurons demonstrated the existence, at the neuronal surface, of heparan sulfate-specific and saturable binding sites, suggesting that SR 80037A activates specific signal transduction pathways. In the present study, using rat or mouse neurons from the embryonic cortex, we show that SR 80037A signaling involves one or several G-coupled receptor or receptors, small GTPases rhoA and/or rhoC, and one or several PKCs. We also demonstrate that the rapid soma rounding elicited by SR 80037A does not require protein synthesis but that the long-term effect on dendrite initiation requires protein synthesis in a short period after the addition of the heparan sulfate. Finally, by preparing membranes from the somatodendritic or axonal compartments we demonstrate that the identified signaling pathway is activated by SR 80037A primarily in the somatodendritic compartment and is not sensitive to the addition of a dermatan sulfate glycosaminoglycan that does not induce the axonal phenotype by impairing dendrite initiation and elongation.

Adenosine Diphosphate↗
PubMed1998

Stimulation of mitogenesis by a cell-permeable PI 3-kinase binding peptide.

The binding of small phosphopeptides to the SH2 domains of the p85 regulatory subunit of PI 3-kinase can activate the enzyme in vitro. In the present study a cell-permeable peptide that binds specifically to the SH2 domains of p85 has been evaluated for its ability to stimulate a mitogenic response in the C2 muscle cell line. This peptide, in contrast to four other SH2-binding peptides, was as effective as serum, EGF, and FGF at stimulating entry into S-phase. The response to the p85 binding peptide, but not FGF, was inhibited by wortmannin and rapamycin, indicating that the peptide activates the PI 3-kinase/S6 kinase signalling pathway. The peptide response was not inhibited by the MEK inhibitor (PD098059) and did not stimulate Erk phosphorylation. Thus, there would appear to be no direct cross-talk between the pathway activated by the p85 binding peptide and the p42/p44 MAPK cascade.

3T3 Cells↗
PubMed1998

Neural cell adhesion molecule L1: relating disease to function.

Neural cell adhesion molecules of the immunoglobulin superfamily are important components of the network of guidance cues and receptors that govern axon growth and guidance during development. For neural cell adhesion molecule L1, the combined application of human genetics, knockout mouse technology, and cell biology is providing fundamental insight into the role of L1 in mediating neuronal differentiation. Disease-causing mutations as well as mouse models of L1 disruption can now be used to examine the relevance of L1 binding specificities and signal transduction pathways that have been observed in vitro.

Animals↗
PubMed1998

Structural mosaicism on the submicron scale in the plasma membrane.

The lateral mobility of the neural cell adhesion molecule (NCAM) was examined using single particle tracking (SPT). Various isoforms of human NCAM, differing in their ectodomain, their membrane anchorage mode, or the size of their cytoplasmic domain, were expressed in National Institutes of Health 3T3 cells and C2C12 muscle cells. On a 6.6-s time scale, SPT measurements on both transmembrane and glycosylphosphatidylinositol (GPI) anchored isoforms of NCAM expressed in 3T3 cells could be classified into mobile (Brownian diffusion), slow diffusion, corralled diffusion, and immobile subpopulations. On a 90-s time scale, SPT studies in C2C12 cells revealed that 40-60% of transfected NCAM was mobile, whereas a smaller fraction (approximately 10-30%) experienced much slower diffusion. In addition, a fraction of approximately 30% of both transfected GPI and transmembrane isoforms and endogenous NCAM isoforms in C2C12 cells experienced transient confinement for approximately 8 s within regions of approximately 300-nm diameter. Diffusion within both these and the slow diffusion regions was anomalous, consistent with movements through a dense field of obstacles, whereas diffusion outside these regions was normal. Thus the membrane appears as a mosaic containing regions that permit free diffusion as well as regions in which NCAM is transiently confined to small or more extended domains. These results, including a large, freely diffusing fraction, similar confinement of transmembrane and GPI isoforms, a significant slowly diffusing fraction, and relatively large interdomain distances, are at some variance with the membrane skeleton fence model (Kusumi and Sako, 1996). Possible revisions to the model that incorporate these data are discussed.

3T3 Cells↗
PubMed1998

Semisynthetic resorbable materials from hyaluronan esterification.

In recent years, research on new, biocompatible, degradable materials has seen the development of a series of modified natural polymers. Among these, a new class of materials consisting of different hyaluronan derivatives promises to be useful in a whole range of clinical applications thanks to their varied biological properties. These new materials are obtained by chemical modification of purified hyaluronan consisting of the partial or total esterification of the carboxyl groups of this natural polymer. This review on the properties of the new materials reports some of their biocompatibility and characterization aspects based on findings from studies conducted on the ethyl and benzyl hyaluronan esters, two representative members of this new class of compounds, and is intended to arouse interest in the potential of other, as yet unexplored derivatives. From the results of a number of investigations, the various derivatives appear to possess different physico-chemical properties, especially as far as the degree of hydration and polymer stability are concerned. In addition, the type of esterification and extent of chemical esterification of hyaluronan considerably affects the biological properties of these materials, offering a range of polymers either favouring or, conversely, inhibiting the adhesion of certain types of cell.

Absorbable Implants↗
PubMed1998

Automated system for the on-line monitoring of powder blending processes using near-infrared spectroscopy. Part II. Qualitative approaches to blend evaluation.

Near Infrared (NIR) spectroscopy is seen as a very powerful tool in a variety of applications involving powder characterisation. Here we deal with a typical pharmaceutical application of powder blend monitoring. A D-optimal experimental design is used to cover the 85-115% range of the target formulation which is comprised of the active component at 3.5% w/w, Microcrystalline Cellulose (Avicel PH102) at 62%, Dibasic Calcium Phosphate Anhydrous at 31.5%, Sodium Starch Glycolate (Explotab) at 2%, and 1% Magnesium Stearate. A miniature Flobin blender has been modified to enable the use of a fibre optic probe for on-line NIR spectral data collection. The experiments were successful in detecting spectral changes which eventually converged to constant variance. While the NIR spectrum of a powdered sample is rich in information which is representative of both the physical and chemical characteristics of the sample, it is at times difficult to select the appropriate mathematical treatments in order to extract the desired information. This article investigates several possible pre-treatments (including detrending (DT), standard normal variates (SNV), second derivatives, and the combination of SNV and DT) together with several ways in establishing blend homogeneity, which includes the running block standard deviation, dissimilarity calculations and principal components analysis (PCA). The focus of this work is to investigate qualitative tools of analysis for blend homogeneity determinations, while future work will focus on quantitative data interpretation.

Algorithms↗
PubMed1998

A novel approach for inhibiting growth factor signalling in murine tooth development. Inhibition of FGF's.

Growth factors belonging to the FGF and TGF-beta families, together with other secreted factors such as Sonic hedgehog, have been shown to be spatially and temporally regulated during tooth development. Providing evidence of the functions of these molecules has, however, proved difficult. We have developed a novel strategy for investigating the role of secreted molecules in tooth development using soluble forms of membrane bound receptors to sequester ligands. Chimeric fusion proteins of receptor extracellular domains were cloned into the eukaryotic expression vector pIG-1 and transfected into COS cells. Fusion proteins secreted by the COS cells were purified using Protein A Sepharose. A soluble form of the FGF receptor FGF-1IIIc, which preferentially binds FGF-2 and FGF-4, was produced using this technique and added to mouse mandible cultures. Addition of the soluble receptors to E13 cultures resulted in down-regulation of Sonic hedgehog expression in molar enamel knots, consistent with inhibition of FGF-4 signalling.

Animals↗