Defective arterial monitoring kits.
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Blood Pressure Monitors↗
Biomedical subjects
P Doherty
Publications and source records attributed to P Doherty.
Cellular determinants of the lateral mobility of neural cell adhesion molecules.
The lateral mobility of the neural cell adhesion molecule (NCAM) was examined using fluorescence recovery after photobleaching (FRAP). Various isoforms of human NCAM, differing in their ectodomain, their membrane anchorage mode or in the size of their cytoplasmic domain, were expressed in NIH 3T3 cells and C2C12 muscle cells. When the various isoforms were compared in 3T3 cells, FRAP studies showed both GPI-anchored and transmembrane isoforms diffused rapidly and only small differences in either the diffusion coefficients (D) or the mobile fractions (mf) were measured, suggesting the importance of the ectodomain in regulating lateral diffusion. However, the mobility of all NCAM isoforms was greatly reduced in regions of cell-cell contact, presumably due to homophilic trans interactions between NCAMs on adjacent cells. NCAM isoforms transfected into C2C12 cells which express NCAM naturally usually displayed a significantly lower D compared to the same isoforms transfected into 3T3 cells. Thus, NCAM lateral mobility is modulated in regions where cells interact and by the structure of the host cell membrane.
3T3 Cells↗
Selective inhibition of growth factor-stimulated mitogenesis by a cell-permeable Grb2-binding peptide.
The activation of the mitogen-activated protein kinase (MAPK) cascade by a variety of growth factors and other agents is central to a mitogenic response. In the case of polypeptide growth factors such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), the steps leading to activation of MAPK require the function of the adaptor protein Grb2 (growth factor receptor binding protein 2), which can bind either directly or indirectly via its Src homology 2 domain to activated receptor tyrosine kinases. A cell-permeable mimetic of the EGF receptor Grb2 binding site has been investigated for its ability to inhibit biological responses stimulated by a variety of growth factors. Pretreatment of cells with this peptide results in the accumulation of the peptide in cells and its association with Grb2. This is associated with a complete inhibition of the mitogenic response stimulated by EGF and PDGF. In contrast, the peptide has no effect on the mitogenic response stimulated by fibroblast growth factor. The peptide could also inhibit the phosphorylation of MAPK stimulated with EGF and PDGF in the absence of an effect on the fibroblast growth factor response. These data demonstrate that cell-permeable mimetics of Src homology 2 binding sites can selectively inhibit growth factor-stimulated mitogenesis, and also directly demonstrate specificity in the coupling of activated receptor tyrosine kinases to the MAPK cascade.
Adaptor Proteins, Signal Transducing↗
Soluble myelin-associated glycoprotein (MAG) found in vivo inhibits axonal regeneration.
Myelin-associated glycoprotein (MAG) is a potent inhibitor of axonal regeneration when used as a substrate for growth. However, to be characterized definitively as inhibitory rather than nonpermissive, MAG must also inhibit axonal regeneration when presented in solution. Here, we show that soluble dMAG (extracellular domain only), released in abundance from myelin and found in vivo and chimeric MAG-Fc, can potently inhibit axonal regeneration. For both dMAG and MAG-Fc, inhibition is dose-dependent. If myelin-conditioned medium is immunodepleted of dMAG, or if a MAG antibody is included with MAG-Fc, inhibition is completely neutralized. Together with MAG's ability to induce growth cone collapse, these results demonstrate that MAG is an inhibitory molecule and not merely nonpermissive. The results also suggest that MAG binds to a specific receptor and initiates a signal transduction cascade to effect inhibition. Importantly, these results indicate that soluble dMAG detected in vivo could contribute to the lack of regeneration in the mammalian CNS after injury.
Animals↗
Structural features of collapsin required for biological activity and distribution of binding sites in the developing chick.
We have used Fc-chimeras of collapsin-1/Sema III to study the structure-function activity of this recently identified repulsive axonal guidance molecule and to map the distribution of its binding sites during chick development. Our results show that the biological activity of the collapsin-Fc in an in vitro collapse assay is independent of both the Ig-domain and the positive charged carboxy terminus. Collapsin binding sites were found on a number of neuronal fiber tracts, and in two instances (DRG tracts and the retinotectal projection) this expression is both highly dynamic and consistent with them playing a role in axonal growth and guidance. Collapsin-1 binding sites were also found on a number of nonneuronal structures that do not produce collapsin-1 mRNA. We postulate that these sites may act to localize or concentrate collapsin-1 released from growing axons and in this way allow for an autocrine axonal guidance mechanism to function during development.
Animals↗
Falsely reassuring pulse oximetry in the presence of severe hypoxia.
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False Negative Reactions↗
CAMs and the FGF receptor: an interacting role in axonal growth.
We review the effects of three cell adhesion molecules (NCAM, N-cadherin and L1) on axonal growth responses and discuss evidence that they activate fibroblast growth factor receptors in neurons.
Animals↗
Expression of a dominant negative FGF receptor inhibits axonal growth and FGF receptor phosphorylation stimulated by CAMs.
The cell adhesion molecules (CAMs) NCAM, N-cadherin, and L1 are homophilic binding molecules that stimulate axonal growth. We have postulated that the above CAMs can stimulate this response by activating the fibroblast growth factor receptor (FGFR) in neurons. In the present study, we demonstrate that activation of NCAM and L1 can lead to phosphorylation of the FGFR. Both this and the neurite outgrowth response stimulated by all three of the above CAMs are lost when a kinase-deleted, dominant negative form of FGFR1 is expressed in PC12 cells. In addition, we have generated transgenic mice that express the dominant negative FGFR under control of the neuron-specific enolase (NSE) promoter. We show that cerebellar neurons isolated from these mice have also lost their ability to respond to NCAM, N-cadherin, and L1. A peptide inhibitor of phospholipase C gamma (PLCgamma) that inhibits neurite outgrowth stimulated by FGF also inhibited neurite outgrowth stimulated by the CAMs. Thus, we conclude that activation of the FGFR is both necessary and sufficient to account for the ability of the above CAMs to stimulate axonal growth, and that PLCgamma is a key downstream effector of this response.
3T3 Cells↗
Neural cell adhesion molecules of the immunoglobulin superfamily: role in axon growth and guidance.
NCAM, L1, and DCC--immunoglobulin cell adhesion molecules (Ig CAMs)--are widely expressed during development. Many workers have dismissed a role for such molecules in the control of axonal growth and guidance because they do not show highly restricted expression patterns. Yet evidence from a number of model systems suggests all three CAMs play a role in the development of specific projections in the nervous system. For example, there is a reduction in mossy fiber tracts in the hippocampus of mice that lack NCAM, a requirement for DCC in the response of commissural neurons to a floor plate-derived chemoattractant, and a loss of corticospinal tracts in humans who carry mutations in the L1 gene. The above paradox might be explained by the observation that differential post-translational processing can modulate CAMs function and that alternative splicing can generate functionally distinct isoforms of a CAM. Activation of the FGF tyrosine kinase receptor is required for the responses stimulated by NCAM and L1, and the importance of regulated tyrosine phosphorylation for growth and guidance is underscored by the involvement of receptor tyrosine phosphatases in this process.
Animals↗
Cell signalling and CAM-mediated neurite outgrowth.
A wide range of molecules promote nerve growth, and these include cell adhesion molecules (CAMs), NCAM, N-cadherin, and the L1 glycoprotein are CAMs that are normally found on both the advancing growth cone and also on cellular substrates, and in general operate via a homophilic binding mechanism. In recent years it has become clear that nerve growth stimulated by these CAMs does not rely on the adhesion function of these molecules, but instead requires that the CAMs activate second messenger cascades in neurons. A large body of evidence supports the hypothesis that homophilic binding of the CAM in the substrate to the CAM in the neuron leads to activation of the neuronal FGF receptor, possibly via a direct interaction in cis between the CAM and the FGF receptor. The consequential activation of PLC gamma is both necessary and sufficient to account for the neurite outgrowth response stimulated by the above three CAMs. Based on the above model, we reasoned that soluble CAMs might also be able to stimulate neurite outgrowth and that such agents might be developed as potential therapeutic agents for stimulating nerve regeneration. To this end we have made soluble chimeric molecules consisting of the extracellular domain of NCAM or L1 fused to the Fc region of human IgG 1. We have found that these molecules can stimulate neurite outgrowth from rat and mouse cerebellar granule cells cultured on a variety of tissue culture substrates and that they do so by activating the FGF receptor signal transduction cascade in the neurons. Consistent with this model, we find that neurons that have their FGF receptor function ablated as a consequence of the expression of dominant negative FGF receptors, no longer respond to the soluble CAM. Downstream targets of CAM function have also been studied. Addition of soluble CAMs to isolated growth cone preparations from mouse or rat brain leads to enhanced phosphorylation of the GAP-43 protein providing a link between the cell surface and the cytoskeleton.
Animals↗
Signal transduction mechanisms underlying axonal growth responses stimulated by cell adhesion molecules.
The mechanisms underlying nerve growth have been extensively studied, and it has been found that the three cell adhesion molecules (CAMs) L1, NCAM and N-cadherin play a key role in this process. All three CAMs are able to stimulate axonal growth from a variety of neuronal cells, and a range of agents which either mimic or inhibit CAM stimulated neurite outgrowth have been identified and has provided a basis for understanding the nature of the response. Results from these studies suggested that activation of a tyrosine kinase-phospholipase C gamma (PLC gamma) cascade was required for the CAM response. Following the identification of a CAM-homology domain (CHD) within the fibroblast growth factor receptor (FGFR) and a putative CHD-binding motif within each of the CAMs, it was suggested that this might be the tyrosine kinase implicated in the CAM pathway. This has been tested experimentally in a number of ways, including the use of transgenic mice expressing a dominant-negative FGFR, and several results have now demonstrated that a functional FGFR is required for CAM stimulated neurite outgrowth. More recently, treatment of neurons with the CAMs has been shown to stimulate FGFR autophosphorylation and PLC gamma activity which in turn leads to activation of a second messenger cascade involving diacylglycerol and arachidonic acid and results in calcium influx into the neurons. Pharmacological studies have confirmed that this cascade is responsible for the neurite outgrowth response.
Animals↗
Elucidation of the molecular actions of NCAM and structurally related cell adhesion molecules.
The Neural Cell Adhesion Molecule (NCAM) is a founder member of a large family of cell surface glycoproteins that share structural motifs related to immunoglobulin and fibronectin type III (FN III) domains [Walsh and Doherty (1991) (Fig. 1). These glycoproteins have been grouped based on the respective number of each type of domain. In vertebrates members of this family of glycoproteins include L1/NILE, NgCAM, axonin-1/TAG-1, and Thy-1 as well as NCAM. In addition structural homologs of NCAM and L1 have been identified in Drosophila and Grasshoppers [Walsh and Doherty (1991)]. These insect homologs are called fasciclins and a series of mutants corresponding to these aspects of synaptic plasticity [Mayford et al. (1992) Science 256:638-644]. In vertebrates all of these glycoproteins are expressed in the developing nervous system where they have been identified as candidate molecules for mediating axon outgrowth, fasciculation, regeneration, and target recognition. In addition, NCAM is expressed in a number of different tissues and cell types. For example, NCAM is expressed in a dynamic pattern in developing and regenerating adult muscle. In this review we aim to describe important aspects of the role of these CAMS in development of the nervous system, including the neuromuscular junction. Furthermore, we will explore the prospective use of molecular biology, cell biology, and molecular genetic techniques, such as transgenic mice, to understand the role and molecular action of this family of cell adhesion molecules in vivo.
Amino Acid Sequence↗
Inhibition of FGF-stimulated phosphatidylinositol hydrolysis and neurite outgrowth by a cell-membrane permeable phosphopeptide.
BACKGROUND: Activated receptor tyrosine kinases bind downstream effector molecules with high affinity. Provided that they can be introduced into cells, peptides corresponding to these high-affinity sites should be able to compete for the interaction and thereby inhibit specific signal transduction cascades. The high-affinity binding site for phospholipase C gamma (PLCgamma) on the activated fibroblast growth factor receptor (FGFR) is centred around the tyrosine at position 766 (766Tyr), and peptides corresponding to this site inhibit PLCgamma binding to the receptor in vitro. A 16 amino-acid peptide from the third helix of the Antennapedia homeodomain protein has recently been shown to be able to act as an internalization vector that can deliver other peptides into cells. Here, we have designed a peptide that contains both the internalization sequence and the FGFR high-affinity binding site for PLCgamma, and tested it in cultures of cerebellar neurons for its ability to inhibit the activation of PLCgamma by basic FGF. RESULTS: The peptide containing the FGFR high-affinity binding site for PLCgamma inhibited phospholipid hydrolysis stimulated by basic FGF with a maximal effect at 1 microg ml-1. Phosphorylation of 766Tyr was required for this effect. The phosphorylated peptide had no effect on phospholipid hydrolysis stimulated by platelet-derived growth factor, neurotrophin-3 and bradykinin. The phosphorylated peptide also inhibited neurite outgrowth stimulated by FGF, but had no effect on neurite outgrowth stimulated by agents that activate the FGFR signal transduction cascade downstream from the activation of PLCgamma. CONCLUSIONS: Cell-permeable peptides can be designed that inhibit the function of receptor tyrosine kinases. In this context we have developed a peptide that prevents the FGFR from activating PLCgamma, and have used this peptide to obtain the first direct evidence that activation of PLCgamma is required for the neurite outgrowth response stimulated by basic FGF.
Amino Acid Sequence↗
Promiscuity of fibroblast growth factor receptors.
Fibroblast growth factor receptors (FGFRs) have been implicated in many developmental and regenerative events, including axial organisation, mesodermal patterning, keratinocyte organisation and brain development. The consensus view that this reflects a role for one or other of the nine known members of the fibroblast growth factor family in these processes has recently been challenged by the suggestion that FGFRs might be directly activated by a much wider range of ligands, including heparan sulphate proteoglycans and neural cell adhesion molecules. In addition, two novel soluble ligands for FGFRs have been identified using yeast two-hybrid technology. Overall, the new findings suggest that in terms of ligand binding the FGFRs might be an even more promiscuous family of receptor tyrosine kinases than was already appreciated.
Amino Acid Sequence↗
CAM-FGF receptor interactions: a model for axonal growth.
A number of experimental paradigms have been used to demonstrate that NCAM, N-cadherin, and L1 stimulate axonal growth. The molecular basis of this response has been extensively studied and a range of agents that inhibit neurite outgrowth stimulated by the above CAMs, but not integrins, have now been identified. These studies pointed to the activation of a tyrosine kinase-PLCgamma cascade as being important for the neurite outgrowth responses stimulated by all three CAMs, and this was substantiated by the identification of agents that could activate the cascade and mimic the growth response. In this review we will suggest that the neurite growth response stimulated by these CAMs is mediated by activation of the fibroblast growth factor receptor (FGFR) in neurons and that this results in the recruitment and activation of PLCgamma via interactions of its SH2 domain with the activated receptor. In this context the key events downstream from activation of PLCgamma required for neurite growth appear to be the conversion of diacylglycerol (DAG) to arachidonic acid (AA) via DAG lipase activity, followed by an increased influx of calcium into the neurons. The evolutionary conservation of putative binding motifs between the above CAMs and the FGFR suggests that activation of the FGFR-PLCgamma cascade by the CAMs might involve a direct CAM-FGFR interaction. The identification of the binding motifs also allows for predictions to be made concerning whether other CAMs might directly interact with the FGFR.
Amino Acid Sequence↗
Fibroblast growth factor receptor function is required for the orderly projection of ganglion cell axons in the developing mammalian retina.
During the early stages of development various cell adhesion molecules (CAMs) and fibroblast growth factor receptors (FGFR) are expressed throughout the retinal neuroepithelium. The ability of retinal ganglion cells to project their axons to the optic fissure depends, in part, on cell-cell interactions mediated by cell adhesion molecules. In the present study we show that the ability of the firstborn rat retinal ganglion cells to extend axons in vitro can be stimulated by NCAM and L1, but not N-cadherin. Both CAM responses can be fully inhibited by antibodies that block neuronal fibroblast growth factor receptor function and by agents that block defined steps in the FGFR signal transduction cascade. When added to living E13.5 rat retinal whole-mount preparations the same agents induced errors in the orderly establishment of young axon patterns in the retinal periphery and caused axons in the retinal center to defasciculate. These results suggest that the activation of the fibroblast growth factor receptor signal cascade not only promotes survival and proliferation of various cell types but can also mediate intraretinal axon guidance.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Pain relief with low-dose intravenous clonidine in a child with severe burns.
The case of an 11-year-old boy who suffered second and third degree burns to 78% of his body is reported. The large doses of morphine used as analgesia resulted in severe side effects: ventilatory dependence, impairment of gastrointestinal function and psychological disturbance. Intravenous lignocaine was added without benefit. The addition of low-dose intravenous clonidine, however, precipitated a dramatic reduction in morphine consumption with an attendant improvement in ventilatory, gastrointestinal and psychological functions.
Adrenergic alpha-Agonists↗
Automated system for the on-line monitoring of powder blending processes using near-infrared spectroscopy. Part I. System development and control.
An automated system for the on-line monitoring of powder blending processes is described. The system employs near-infrared (NIR) spectroscopy using fibre-optics and a graphical user interface (GUI) developed in the LabVIEW environment. The complete supervisory control and data analysis (SCADA) software controls blender and spectrophotometer operation and performs statistical spectral data analysis in real time. A data analysis routine using standard deviation is described to demonstrate an approach to the real-time determination of blend homogeneity.
Chemistry, Pharmaceutical↗