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E J Williams

Publications and source records attributed to E J Williams.

At least 55 records · Page 3Linked to original sources
PubMed1997

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↗
PubMed1996

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↗
PubMed1996

Use of subvoxel registration and subtraction to improve demonstration of contrast enhancement in MRI of the brain.

To assess the potential of registration of images before and after contrast medium for improving the demonstration of contrast enhancement, we compared conventional 2D T1-weighted spin-echo images with precisely registered 3D volume images and subtraction images derived from them in 2 normal subjects and 30 patients with a variety of brain disease. The volume images were registered to subvoxel accuracy using a rigid body translation and rotation, sinc interpolation and a least-squares fit; subtraction images were obtained from these. Normal contrast enhancement was demonstrated better with positionally registered volume and subtraction images than with conventional images in the meninges, ependyma, diploic veins, scalp, skin, orbit and sinuses. Abnormal enhancement was seen better in meningeal disease, multiple sclerosis and tumours as well as on follow-up studies. Subvoxel registration of images before and after contrast medium may be of considerable value in the recognition of contrast enhancement where there are small changes, or where the changes affect tissues with high or low baseline signal values. The technique also appears likely to be of value in demonstrating contrast enhancement in tissues at interfaces and at other areas of complex anatomy, and in follow-up studies.

Brain↗
PubMed1996

Assessment of brain changes with registered MR before and after bone marrow transplantation for chronic myeloid leukemia.

PURPOSE: To determine the frequency and nature of changes to the brain resulting from chemotherapy, radiation therapy, and bone marrow transplantation for chronic myeloid leukemia and to compare the sensitivity of conventional and registered MR scans for detecting these changes. METHODS: In 15 patients, conventional T1-weighted, T2-weighted, and fluid-attenuated inversion recovery MR sequences, as well as T1-weighted radio frequency spoiled 3-D volume MR scans were performed before, 4 to 6 days after, and up to 339 days after transplantation (13 allografts, two autografts). A subvoxel registration program was used to match the volume images precisely so that small changes could be detected after subtraction of scans. Five healthy adult control subjects were also studied on two occasions 1 month apart. RESULTS: Studies performed 4 to 339 days after transplantation showed ventricular enlargement and cortical atrophy in all 13 patients who had allografts. The changes were evident at 4 to 6 days after transplantation and became more obvious during later follow-up examinations. Similar changes were seen in one patient with an autograft but no significant change was seen in the other patient with an autograft or in the five control subjects. Accurately registered volume scans were more sensitive than unregistered conventional scans in detecting early (9/10 versus 0/10), intermediate (12/13 versus 3/12), and late (10/10 versus 4/9) ventricular enlargement on follow-up examinations. The same applied to cortical atrophy (9/10 versus 0/10, 12/13 versus 0/12, and 10/10 versus 0/9). CONCLUSION: The specific cause and clinical significance of these changes are uncertain. Subvoxel registration of serial MR images may reveal changes that are poorly seen or not apparent on conventional scans.

Adolescent↗
PubMed1995

A Ca2+/calmodulin kinase inhibitor, KN-62, inhibits neurite outgrowth stimulated by CAMs and FGF.

We have used monolayers of parental 3T3 fibroblasts and 3T3 cells expressing transfected cell adhesion molecules (CAMs, NCAM, N-cadherin, or L1) as a culture substrate for cerebellar neurons. Previous studies suggest that the transfected CAMs promote neurite outgrowth by activating a second messenger pathway within the responding neuron that involves influx of calcium into neurons as a consequence of activation of an FGF receptor. The same neurite outgrowth response can be induced by FGF or a number of agents that directly activate defined steps in the CAM signaling pathway. In the present study we show that the neurite outgrowth stimulated by the above three CAMs, FGF, arachidonic acid (AA), and K+ depolarization can be abolished by the Ca2+/calmodulin-dependent (CaM) kinase inhibitor, KN-62. We also demonstrate that neurite outgrowth over astrocytes, which represent a more physiologically relevant cellular substrate, can be substantially inhibited by a number of agents that block the CAM signaling pathway, including KN-62. However, neurite outgrowth induced by activation of protein kinase A is unaffected by inhibition of CaM kinase activity as is basal neurite outgrowth over 3T3 monolayers or a polylysine/laminin substrate. These results suggest that CaM kinase activity is specifically required downstream of calcium influx in the CAM and FGF signaling pathway leading to axonal growth.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Antigliadin antibody measurement by chemiluminescence ELISA in the diagnosis of coeliac disease.

AIMS: To develop a chemiluminescence enzyme linked immunosorbent assay (ELISA) for the detection of circulating gliadin antibodies in the diagnosis of coeliac disease. To compare this method for linearity and sensitivity with an established colorimetric method. METHODS: Three sets of age and sex matched patient groups were studied: normal controls (patients with no clinical signs of intestinal disorders); gastrointestinal controls (patients with a known gastrointestinal disorder other than coeliac disease); and patients in whom suspected coeliac disease had been confirmed by positive jejunal biopsy. IgG antigliadin antibody (IgG-AGA) and IgA antigliadin antibody (IgA-AGA) titres were determined. RESULTS: Comparison of the colorimetric and chemiluminescence methods showed close correlation of measured antibody levels for both control patient groups. In the coeliac patients correlation of antibody levels measured by both methods was not possible because the colorimetric assay is limited by the spectrophotometer's limits of detection. This problem was overcome by the chemiluminescence method which was linear over a greater range and to far higher values. CONCLUSIONS: The chemiluminescence ELISA performs as well as the colorimetric assay at low and average antibody levels and has the advantage of also giving a numerical value to higher antibody titres. The method was accurate and reproducible in confirming the diagnosis of coeliac disease in patients with positive jejunal biopsy and was capable of monitoring progress of the disease.

Adolescent↗
PubMed1995

FGF inhibits neurite outgrowth over monolayers of astrocytes and fibroblasts expressing transfected cell adhesion molecules.

We have cultured cerebellar neurons on monolayers of cortical astrocytes in control medium or medium containing recombinant basic fibroblast growth factor (FGF). FGF was found to inhibit neurite outgrowth, with a significant effect seen at 0.5 ng/ml and a maximal effect at 10 ng/ml. FGF increased the production of arachidonic acid (AA) in cerebellar neurons, and when added directly to cultures or generated endogenously via activation of phospholipase A2 using melittin, this second messenger could mimic the inhibitory effect of FGF. FGF and AA could also specifically inhibit neurite outgrowth stimulated by three cell adhesion molecules (NCAM, N-cadherin and L1) expressed in transfected fibroblasts, or in the case of L1 bound to a tissue culture substratum. These data demonstrate that, in certain cellular contexts, FGF can act as an inhibitory cue for axonal growth and that arachidonic acid is the second messenger responsible for this activity. We discuss the possibility that arachidonic acid inhibits neurite outgrowth by desensitising the second messenger pathway underlying neuronal responsiveness to cell adhesion molecules.

3T3 Cells↗
PubMed1994

Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin.

Cell contact-dependent neurite outgrowth stimulated by CAMs requires activation of a second messenger pathway that requires the function of a tyrosine kinase upstream from calcium influx into neurons. In the present study, we present evidence that implicates activation of the fibroblast growth factor receptor (FGFR) in the pathway underlying neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. We have identified a CAM homology domain in the FGF family of receptors and show that antibodies which bind to this domain specifically inhibit neurite outgrowth stimulated by the above CAMs. We also show that synthetic peptides derived from this domain can differentially and specifically inhibit neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. In addition, a soluble L1-Fc chimera is shown to stimulate an increase in phosphotyrosine on the same set of neuronal proteins that are phosphorylated following activation of the FGFR with basic FGF.

Amino Acid Sequence↗
PubMed1994

The production of arachidonic acid can account for calcium channel activation in the second messenger pathway underlying neurite outgrowth stimulated by NCAM, N-cadherin, and L1.

We have used monolayers of control 3T3 fibroblasts and 3T3 fibroblasts expressing transfected cell adhesion molecules (CAMs)--NCAM, N-cadherin, and L1--as a culture substrate for cerebellar neurones. The transfected CAMs promote neurite outgrowth by activating a second messenger pathway that culminates in calcium influx into neurones through N- and L-type calcium channels. We show that the same neurite outgrowth response can be directly induced by arachidonic acid (10 microM) and that this response can be inhibited by N- and L-type calcium channel antagonists. In cells, arachidonic acid can be generated by phospholipase A2 or by the sequential activities of a phospholipase C (to generate diacylglycerol) and diacylglycerol lipase. In the present study we show the neurite outgrowth stimulated by CAMs (but not by various other agents) can be abolished by an inhibitor of diacylglycerol lipase acting at a site upstream from calcium channel activation. The results suggest that arachidonic acid and/or one of its metabolites is the second messenger that activates calcium channels in the CAM signalling pathway leading to axonal growth, and this is supported by recent evidence that shows the same concentrations of arachidonic acid can increase voltage-dependent calcium currents in cardiac myocytes.

3T3 Cells↗
PubMed1994

Neurite outgrowth stimulated by the tyrosine kinase inhibitor herbimycin A requires activation of tyrosine kinases and protein kinase C.

Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events down-stream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N- and L-type calcium channels.

Animals↗
PubMed1994

Tyrosine kinase inhibitors can differentially inhibit integrin-dependent and CAM-stimulated neurite outgrowth.

We have used monolayers of parental 3T3 cells and 3T3 cells expressing one of three transfected cell adhesion molecules (CAMs) (NCAM, N-cadherin, and L1) as a culture substrate for rat cerebellar neurons. A number of tyrosine kinase inhibitors have been tested for their ability to inhibit neurite outgrowth over parental 3T3 monolayers which we show to be partly dependent on neuronal integrin receptor function, as compared with neurite outgrowth stimulated by the above three CAMs. Whereas genistein (100 microM), lavendustin A (20 microM), and tyrphostins 34 and 47 (both at 150 microM) had no effect on integrin dependent or CAM stimulated neurite outgrowth, the erbstatin analogue (10-15 micrograms/ml) and tyrphostins 23 and 25 (both at 150 microM) specifically inhibited the response stimulated by all three CAMs. CAM stimulated neurite outgrowth can be accounted for by a G-protein-dependent activation of neuronal calcium channels; experiments with agents that directly activate this pathway localized the erbstatin analogue site of action upstream of the G-protein and calcium channels, whereas tyrphostins have sites of action downstream from calcium channel activation. These data suggest that activation of an erbstatin sensitive tyrosine kinase is an important step upstream of calcium channel activation in the second messenger pathway underlying the neurite outgrowth response stimulated by a variety of CAMs, and that this kinase is not required for integrin-dependent neurite outgrowth.

3T3 Cells↗
PubMed1994

Characterisation of the second messenger pathway underlying neurite outgrowth stimulated by FGF.

Cerebellar neurons, cultured on monolayers of 3T3 fibroblasts or on a polylysine/laminin-coated substratum, responded to recombinant basic FGF by extending longer neurites. The response was biphasic reaching a maximum at 5 ng/ml FGF, but desensitising at 100-200 ng/ml FGF. The response to FGF could be inhibited by a tyrosine kinase inhibitor (the erbstatin analogue), by a diacylglycerol lipase inhibitor (RHC-80267) and by a combination of N- and L-type calcium channel antagonists or other agents that negate the effects of calcium influx into neurons. The response to FGF could be fully mimicked by arachidonic acid added directly to the cultures, or generated via activation of phospholipase A2 with melittin. The response to melittin, but not to FGF or arachidonic acid, was inhibited by 4-bromophenacyl bromide, a phospholipase A2 inhibitor. The response to arachidonic acid was also biphasic and high concentrations of this agent could cross-desensitise the FGF response and vice versa. The response to arachidonic acid could be fully inhibited by the agents that block or negate the effects of calcium influx into neurons, but was not inhibited by the tyrosine kinase or diacylglycerol lipase inhibitors. These data suggest that FGF stimulates neurite outgrowth by activating a cascade that involves activation of phospholipase C gamma to produce diacylglycerol, conversion of diacylglycerol to arachidonic acid by diacylglycerol lipase and the activation of voltage-gated calcium channels by arachidonic acid.

3T3 Cells↗
PubMed1992

Calcium influx into neurons can solely account for cell contact-dependent neurite outgrowth stimulated by transfected L1.

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.

3T3 Cells↗
PubMed1991

An experiment in medical education. A critical analysis using traditional criteria.

In 1984, in addition to its standard traditional curriculum, Rush Medical College (Chicago, Ill) developed a Socratic problem-based method of teaching basic science material called the alternative curriculum. As part of an evaluation of this new curriculum, students in the two curricula were compared using three traditional measurements: (1) test scores from the National Board of Medical Examiners, Part I; (2) test scores from the National Board of Medical Examiners, Part II; and (3) performance on an oral examination. Alternative curriculum students did not differ significantly from their traditional curriculum classmates on National Board of Medical Examiners, Part I and Part II total scores, although their subset scores on Part I did tend to be lower, reaching significance in one subset area. Differences in performance favoring the traditional curriculum were primarily seen in the early years of the program. Alternative curriculum students in the class matriculated in 1987 scored significantly higher in three of five categories on the oral examination.

Adult↗