Search PubMed⌕ Search

Biomedical subjects

P Doherty

Publications and source records attributed to P Doherty.

At least 127 records · Page 7Linked to original sources
PubMed1989

K-252a specifically inhibits the survival and morphological differentiation of NGF-dependent neurons in primary cultures of human dorsal root ganglia.

We have measured neurofilament protein expression in cultures of human dorsal root ganglia (DRG) grown in both the presence and absence of nerve growth factor (NGF). Cultures established from DRG of 8-13 weeks gestation contained populations of both NGF-dependent and -independent neurofilament-positive cells. A recently described kinase inhibitor named K-252a inhibited NGF-dependent increases in neurofilament protein expression with half-maximal and maximal inhibition apparent at concentrations of 6 and 25 nM. At up to 50 nM, K-252a had no effect on neurofilament protein expression from NGF-independent neurons.

Carbazoles↗
PubMed1989

Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms.

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.

Animals↗
PubMed1989

Increased intracellular cyclic AMP differentially modulates nerve growth factor induction of three neuronal recognition molecules involved in neurite outgrowth.

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.

Antigens, Surface↗
PubMed1988

Factors controlling the expression of the NGF receptor in PC12 cells.

An enzyme-linked immunoadsorbent assay (ELISA) has been used to study the relative expression of the nerve growth factor receptor (NGF-R) in PC12 cells. Both nerve growth factor (NGF) and fibroblast growth factor (FGF) were found to induce time-dependent increases in receptor expression. The former response was half-maximal at approximately 2 ng/ml and maximal at 25-50 ng/ml. The induction of NGF-R expression by NGF was associated with an increase in the relative level of a 3.8 kb mRNA species encoding this protein. Whereas the induction of the NGF-R by NGF and FGF were both fully inhibited by cholera toxin and cordycepin, the responses were differentially inhibited by the kinase inhibitor K-252a.

Animals↗
PubMed1988

Analysis of PC12 cell adhesion to muscle and non-muscle cells and components of the extracellular matrix.

The relative strength of PC12 cell adhesion to monolayers of myoblasts and fibroblasts as well as to purified components of the extracellular matrix has been determined. PC12 cell adhesion to myoblasts and fibroblasts was dependent on temperature, and the relative strength of adhesion to both increased over a 4-h period. The strength of adhesion to myoblasts was consistently found to be greater than that to fibroblasts. Whereas coating tissue culture plastic with purified collagen and laminin increased its ability to support PC12 cell adhesion, coating with plasma fibronectin or gelatin reduced cell adhesion. Trypsin treatment of PC12 cells was without effect on their adhesion to collagen and laminin and only partially reduced the strength of adhesion to myoblasts. In contrast trypsinized cells showed a greatly reduced ability to adhere to fibroblast monolayers. Pretreatment of PC12 cells with NGF had no obvious effect on the ability of the cells to adhere to any of the above substrata.

Adrenal Gland Neoplasms↗
PubMed1988

Comparison of the effects of NGF, activators of protein kinase C, and a calcium ionophore on the expression of Thy-1 and N-CAM in PC12 cell cultures.

The addition of nerve growth factor (NGF) to PC12 cells induces an approximate doubling in the cell surface expression of the Thy-1 glycoprotein and the neural cell adhesion molecule (N-CAM) after 24 h of culture. Although both responses are measured at the same time point, their sensitivity to NGF differed with half-maximal induction of Thy-1 apparent at NGF concentrations (approximately 0.1 ng/ml NGF) that had little effect on N-CAM expression. Phorbol ester derivatives capable of activating Ca2+/phospholipid-dependent protein kinase (protein kinase C) and the calcium ionophore A23187 were found to mimic the NGF induction of Thy-1, but not N-CAM. Similar results were observed when a synthetic diacylglycerol was added to PC12 cell cultures. Increased expression of Thy-1 consequent to phorbol ester, calcium ionophore, or NGF treatment was associated with an increase in the expression of the mRNA species that encodes Thy-1. Increased expression of Thy-1 consequent to all three treatments was also reduced by treatment with the transcription inhibitor cordycepin. Treatment of PC12 cells with high concentrations of phorbol esters was found to inhibit the NGF induction of Thy-1, but not N-CAM. Whereas the above results are consistent with activation of protein kinase C underlying the NGF induction of Thy-1, the same data are not consistent with this pathway being important in the N-CAM response.

Antigens, Surface↗
PubMed1987

Nerve growth factor-induced changes in neural cell adhesion molecule (N-CAM) in PC12 cells.

The effects of nerve growth factor (NGF) on the expression of neural cell adhesion molecule (N-CAM) in PC12 cells were determined. A quantitative immunoassay was used to show that NGF induces a 4- to 5-fold increase in relative N-CAM levels over a 3-day period. This increase could not be mimicked by cholera toxin suggesting that it is not a simple consequence of morphological differentiation. The changes in N-CAM levels induced by NGF were accompanied by changes in N-CAM molecular forms. The 140-kd N-CAM species is the major N-CAM expressed by naive PC12 cells, while NGF-treated cultures express N-CAM species of 180 kd and 140 kd. Northern analysis showed that naive cells express a 6.7-kd N-CAM mRNA species only, while NGF-treated cultures express both a 6.7-kb and a 7.2-kb transcript. As the 6.7-kb and 7.2-kb mRNAs are alternative spliced transcripts of a single gene, this result shows that NGF can activate a neuron-specific splicing mechanism. This is the first description of control of N-CAM expression by a growth factor.

Adrenal Gland Neoplasms↗
PubMed1987

Use of indium-111-labeled OC-125 monoclonal antibody in the detection of ovarian cancer.

This is a preliminary study to evaluate the utility of using the monoclonal antibody (CO-125) labeled with indium-111 to image recurrences of ovarian cancer. This technique has been investigated in 23 patients with ovarian cancer and the results have been compared with blood OC-125 levels, CT scans, and findings at second-look surgery. Following infusion of 1 mg of F(ab')2 fragments (1-2 mCi 111In), quantitative SPECT and planar imaging was obtained daily for 72 hr along with analysis of serum. The nuclear medicine scans of the tumor site recurrences were technically excellent. When compared to second-look laparotomy, there were 2 true negatives, 2 false positives, 14 true positives, and 2 false negatives by nuclear imaging. CT scans correlated less well with surgery, but serum OC-125 levels correlate more closely with nuclear scans and second-look surgery. Those with multiple small metastatic implants showed a pattern of diffuse uptake which increased with time, whereas those with nodal or larger recurrences showed a more focal uptake. The combination of favorable biodistribution and positive images, especially in patients with normal antigen levels and negative CT scans, suggests a role for OC-125 monoclonal antibody imaging in their clinical management. However, further investigation is needed to determine whether nuclear scans can replace second-look surgery. If it can show that enough 111In-labeled antibody accumulates in the tumor site to justify radioimmunotherapy, then 90Y (a pure beta emitter) could be exchanged for 111In. This is potentially a method of radioimmunotherapy for recurrent ovarian carcinoma.

Adult↗
PubMed1987

Cholera toxin and dibutyryl cyclic AMP inhibit the expression of neurofilament protein induced by nerve growth factor in cultures of naive and primed PC12 cells.

The effects of nerve growth factor (NGF), dibutyryl cyclic AMP (db cAMP), and cholera toxin on neurofilament protein expression in cultures of PC12 rat pheochromocytoma cells were examined using an enzyme-linked immunoadsorbent assay (ELISA). Morphological differentiation induced by NGF was associated with up to 30-fold increases in the level of neurofilament protein recognised by monoclonal antibody RT97. A more rapid response was apparent from primed as compared to naive PC12 cells. Cholera toxin and db cAMP both induced morphological differentiation of naive PC12 cells, but failed to promote neurite regeneration from primed cells. Neither response was associated with a significant induction of neurofilament protein. Both cholera toxin and db cAMP, but not B-cholera toxin nor antibodies to the toxin receptor, were found to inhibit the neurofilament protein response induced by NGF. Primed cells were more susceptible to this inhibition, and both cholera toxin and db cAMP inhibited neurite regeneration from these cells. These data suggest that increased intracellular cyclic AMP can suppress the expression of neuronal differentiation antigens induced by NGF, and are consistent with a role for neurofilament protein in promoting or facilitating the formation of a stable neuritic network.

Adrenal Gland Neoplasms↗
PubMed1987

Control of Thy-1 glycoprotein expression in cultures of PC12 cells.

The effects of nerve growth factor (NGF) and cholera toxin on the expression of the Thy-1 glycoprotein were examined in cultures of naive and primed PC12 cells using an enzyme-linked immunoadsorbent assay (ELISA). With primed PC12 cells, NGF induced a rapid increase in Thy-1 expression over a time course similar to that of neurite regeneration, with half-maximal and maximal increases apparent at 0.6 and 6 ng/ml NGF. Cholera toxin and dibutyryl cyclic AMP, but not B-cholera toxin or antibodies to the toxin receptor, were found to inhibit NGF-induced increases in Thy-1. Morphological differentiation of naive PC12 cells induced by NGF, but not cholera toxin, was also associated with increased expression of Thy-1. Despite showing a synergistic effect on morphological differentiation, cholera toxin was again found to inhibit NGF-induced increases in Thy-1 expression in cultures of naive PC12 cells. These data suggest that agents that interact directly or indirectly with adenylate cyclase may regulate the responsiveness of PC12 cells to NGF, and as such modulate the expression of the Thy-1 glycoprotein.

Adrenal Gland Neoplasms↗
PubMed1987

Ganglioside GM1 antibodies and B-cholera toxin bind specifically to embryonic chick dorsal root ganglion neurons but do not modulate neurite regeneration.

Polyclonal antibodies to ganglioside GM1 have been prepared and characterised by direct and competitive enzyme-linked immunoassay. An immunoglobulin fraction was prepared from a rabbit antisera showing high specificity and antibody titre for GM1 relative to the other major brain gangliosides. The anti-GM1 immunoglobulin fraction and B-cholera toxin specifically labelled neurons in primary cultures of embryonic chick dorsal root ganglia and there was a good correlation between the relative increase in binding of anti-GM1 immunoglobulin and B-cholera toxin following neuraminidase treatment of a variety of cell types. At antibody concentrations that show saturable binding to endogenous ganglioside in the neuronal membrane, the anti-GM1 immunoglobulin fraction did not interfere with the nerve growth factor (NGF)-mediated fibre outgrowth and neuronal survival as indexed by measurement of neurofilament protein levels. Similarly, at levels in excess of those shown to stimulate thymocyte proliferation, B-cholera toxin was also without effect. These data are not consistent with GM1 in the neuronal membrane functioning as a receptor molecule for NGF and/or other differentiation factors present in the tissue culture media.

Animals↗
PubMed1987

Cell-cell interactions modulate the responsiveness of PC12 cells to nerve growth factor.

The growth of PC12 cells on a collagen substratum or on monolayers of several non-neuronal cell types was studied by measuring nerve growth factor (NGF)-dependent increases in the expression of a 150 X 10(3) (Mr) neurofilament protein subunit and the membrane glycoprotein Thy-1. Both responses were found to be greatly suppressed in cultures of fibroblasts as compared to the C2 and G8-1 muscle cell lines and the C6 glioma cell line. This suppression was associated with an inhibition of NGF-dependent neuritic outgrowth from PC12 cells grown on fibroblast monolayers. There was no evidence that fibroblasts secrete soluble molecules that directly inhibit these responses or neutralize NGF. In addition, there was no difference in the neurofilament protein response from PC12 cells that had been treated with NGF prior to coculture, and the now primed PC12 cells readily extended axons over fibroblast monolayers. These data demonstrate that cell-cell and/or cell-matrix interactions can modulate biochemical responses to NGF and suggest that responsiveness of neuronal cells to environmental cues is not immutable. Control of the latter may be at the level of expression of receptor molecules for cell-surface- or matrix-associated macromolecules and a similar mechanism operating during development could play a role in growth cone guidance.

Cell Communication↗
PubMed1986

Human skeletal muscle cells synthesise a neuronotrophic factor reactive with spinal neurons.

Retrograde trophic influences originating in the skeletal musculature have been postulated to be involved in regulating survival and differentiation of embryonic motor neurons and reactive terminal sprouting of mature motor fibres. We have previously described the use of a quantitative immunoassay for neurofilament protein to bioassay in vitro the cell-type-specific neuronotrophic activity of nerve growth factor (NGF) on sensory ganglion neurons. In the present study, the effect of media conditioned by adult human muscle cells (MCM) on the in vitro development of chicken spinal neurons has been studied using a similar approach. Significant increases in neurofilament protein levels in 7-day chicken embryonic spinal cord cultures were found with doses of MCM protein as low as 0.4 microgram/ml, with a dose-response relationship yielding maximal and half-maximal effects at 4 and 1 microgram/ml, respectively. Maximal increases in neurofilament protein levels were associated with an approximate two-fold increase in neuronal cell survival. MCM also induced increases in choline acetyltransferase activity in chick spinal cord cultures. In both the absence and presence of NGF, MCM did not increase neurofilament protein expression in primary cultures of sensory neurons.

Animals↗
PubMed1986

Changes in miniature end-plate potentials due to moderate hypertonicity at the frog neuromuscular junction.

The effect of an increase in tonicity on the amplitude and frequency of miniature end-plate potentials (m.e.p.p.s) following superfusion of 50 mM- or 100 mM-sucrose-Ringer solution was determined from intracellular recording at end-plates of the frog cutaneous pectoris muscle, in the absence of drugs and using on-line statistical analysis. An immediate decrease in mean amplitude of the order of 20% control was associated with a marked increase in mean frequency. A delayed increase in mean amplitude, independent of frequency, followed the initial response in the majority of end-plates exposed to 100 mM-sucrose-Ringer solution. The net gain was of the order of 34% and was attained over 20-40 min. This was not reversible over at least 20 min. There was an increase in the variability of m.e.p.p. amplitudes associated with the increase of mean amplitude but this was reversible. A moderate increase in tonicity is considered to induce two kinds of presynaptic changes. First, an immediate increase in the probability of release of smaller quantal packets and secondly, an increased loading of transmitter into vesicles within the readily releasable store.

Action Potentials↗
PubMed1986

Effects of amyotrophic lateral sclerosis serum on cultured chick spinal neurons.

We used a quantitative immunoassay to examine the effects of human serum and immunoglobulins on neurofilament protein expression in cultures of chick spinal neurons. Compared with cultures grown in the presence of serum from healthy controls or patients with other neurologic disorders, ALS serum lowered the level of neurofilament proteins. Effects were similar with or without muscle-derived neurotrophic factors; there was no specificity for motor neurons. No neurotoxic activity was found in immunoglobulin fractions, and there was no evidence of circulating antibodies that might neutralize muscle-derived neurotrophic factors or induce cytolysis of spinal neurons.

Adult↗
PubMed1986

Chronic acalculous gallbladder disease: multiimaging evaluation with clinical-pathologic correlation.

Despite the recent advances in hepatobiliary imaging, the diagnosis of chronic acalculous gallbladder disease remains difficult. A retrospective study was undertaken to assess the value of a multiimaging approach in detecting chronic acalculous gallbladder disease and in predicting which patients would obtain symptomatic relief after cholecystectomy. Of 199 patients with chronic cholecystitis, 26 (13%) had no gallstones. Of these 26, only 17 (65%) had symptoms related to chronic cholecystitis; in the remainder, the histologic diagnosis was made incidentally. After cholecystectomy, 13 (76%) of the 17 symptomatic patients obtained long-term symptomatic relief, while in four, the symptoms recurred. Among patients with histologic changes of chronic cholecystitis, biliary scintigraphy was the most sensitive technique (sensitivity, 89%). The sensitivity of sonography and oral cholecystography was 61.5% and 66%, respectively. However, for identifying symptomatic patients who may obtain long-term symptomatic relief after cholecystectomy, the accuracy of sonography, oral cholecystography, and biliary scintigraphy was 82%, 86%, and 38%, respectively. When two tests were in agreement the accuracy was 88%. For chronic acalculous cholecystitis, more than one study must be performed in order to make the correct diagnosis and to predict good results from cholecystectomy.

Adult↗
PubMed1985

Molecular specificity of ganglioside effects on neurite regeneration of sensory neurons in vitro.

Highly purified preparations of individual gangliosides have been tested for their ability to modulate the survival and morphological differentiation of embryonic chick dorsal root ganglion neurons. When added at 48 h to established cultures of nerve growth factor (NGF)-dependent neurons, all ganglioside species tested increased the expression of neurofilament protein. Poly- and trisialogangliosides were more effective than di- or monosialogangliosides. In contrast, neither NGF nor an antiserum against NGF influenced neurofilament protein expression over this period of culture. In addition, ganglioside-induced expression of neurofilament protein was not inhibited by the anti-NGF serum.

Animals↗