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J MacDermot

Publications and source records attributed to J MacDermot.

At least 73 records · Page 4Linked to original sources
PubMed1987

Pyocyanin and 1-hydroxyphenazine produced by Pseudomonas aeruginosa inhibit the beating of human respiratory cilia in vitro.

Pseudomonas aeruginosa culture filtrates varied in their ability to slow human ciliary beat frequency (7-71%). This activity did not correlate with known virulence factors. However, a close correlation (r = 0.97) existed between ciliary slowing and pigment content. In a prolonged culture, the increase in activity correlated (r = 0.94) with pigment accumulation. Gel filtration of lyophilized filtrate yielded a single peak of activity corresponding to the pigment fraction. Pyocyanin extracted from an active strain, and 1-hydroxyphenazine were purified by high performance liquid chromatography, and characterized by ultraviolet absorbance spectra and mass spectrometry. Both slowed cilia in a dose-dependent manner, and were synthesized and shown to be indistinguishable from the biological compounds. Pyocyanin caused gradual onset of slowing and ultimate widespread ciliostasis with epithelial disruption. 1-hydroxyphenazine caused rapid onset of ciliary slowing associated with dyskinesia and ciliostasis. Pyocyanin assayed within filtrates accounted for a significant proportion of the bioactivity present.

Alginates↗
PubMed1986

Purification and structural analysis of pyocyanin and 1-hydroxyphenazine.

Pyocyanin and related members of the phenazine family are produced by Pseudomonas aeruginosa and have been associated with events of pathophysiological importance. Pyocyanin and its base hydrolysis product 1-hydroxyphenazine were purified to homogeneity by reverse-phase high-pressure liquid chromatography. Their mass spectrometric behaviour was examined with a view to evaluating the use of high-resolution chromatography/mass spectrometry in studying phenazine-mediated effects in man. The molecular mass of naturally derived pyocyanin was determined as 210 Da by thermospray liquid chromatography/mass spectrometry and confirmed by desorption electron-impact mass spectrometry. Mass spectrometric data could not be obtained by fast-atom bombardment or desorption chemical ionisation, techniques commonly used to determine molecular mass of polar or thermally labile species. The thermal lability of underivatised pyocyanin precluded analysis by gas chromatography/mass spectrometry. In contrast to pyocyanin, mass spectrometric data were readily obtained for 1-hydroxyphenazine, using direct probe analysis as well as with gas and liquid chromatography inlet systems.

Chromatography, Gas↗
PubMed1986

The putative prostacyclin receptor antagonist (FCE-22176) is a full agonist on human platelets and NCB-20 cells.

The action of FCE-22176 on prostacyclin receptors of human platelets and the NCB-20 cell line have been examined. FCE-22176 is a full agonist in both systems, mediating activation of adenylate cyclase. The concentration required for half-maximum enzyme activation was 174 nM in platelet membranes and 193 nM in homogenates of NCB-20 cells. The binding of [3H]iloprost to human platelet or NCB-20 cell membranes was inhibited by FCE-22176, and Ki values of 400 and 280 nM were obtained.

Adenylyl Cyclases↗
PubMed1986

Stimulation of IgE sensitized human alveolar macrophages by anti-IgE is unaffected by sodium cromoglycate.

Sodium cromoglycate is an effective prophylactic in the treatment of asthma. However, its mechanism of action is still uncertain. The release of thromboxane B2 and N-acetyl-beta-D-glucosaminidase activity was measured from human alveolar macrophages sensitized with human myeloma IgE. Cells were challenged with rabbit affinity purified anti-IgE (no activity against IgG) in the absence or presence of sodium cromoglycate (10(-4)-10(-8)M). There was no change in the release of either thromboxane B2 or N-acetyl-beta-D-glucosaminidase in the presence of sodium cromoglycate at these concentrations.

Acetylglucosaminidase↗
PubMed1986

Prostaglandin endoperoxide analogues which are both thromboxane receptor antagonists and prostacyclin mimetics.

Two prostaglandin endoperoxide analogues, EP 035 and EP 157, behave as specific thromboxane receptor antagonists on isolated smooth muscle preparations such as rabbit aorta, dog saphenous vein and guinea-pig trachea. However, in human platelet-rich plasma (PRP) they produce an unsurmountable block of aggregation induced by a wide range of agents (ADP, platelet-activating factor, thrombin); this inhibitory profile is typical of that seen with either prostaglandin I2 (PGI2) or PGD2. EP 035 and EP 157 induce large increases in cyclic AMP levels (up to 20 times basal) in human PRP. Simultaneous exposure to PGE1 markedly reduces their effect on cyclic AMP; exposure to PGD2 is much less effective in this respect. The adenylate cyclase inhibitor SQ 22,536 opposes the inhibitory action of EP 035, EP 157, iloprost (a stable PGI2 analogue) and PGD2 on platelet aggregation. However, the xanthone derivative AH 6809 blocks the inhibitory action of PGD2 but does not affect EP 035, EP 157 and PGI2 and its structural analogues. EP 035 and EP 157 displace [3H]-iloprost binding to the PGI2 receptor on human platelet membranes. Displacing ability is ranked as follows: iloprost greater than 6a-carba PGI2 greater than EP 157 greater than EP 035 greater than EP 164 (alpha-dinor derivative of EP 157). This order of potency is the same as that found for activation of adenylate cyclase in homogenates of washed human platelets and for inhibition of aggregation in washed human platelets. The activities of EP 035 and EP 157 were studied in two other systems containing PGI2 receptor-adenylate cyclase complexes, the NCB-20 cell line and human lung tissue. In both cases stimulation of adenylate cyclase was found but maximum rates were below that achieved with iloprost. These effects of EP 035 and EP 157 could be correlated with their abilities to displace [3H]-iloprost binding. 5 These results indicate that EP 035 and EP 157 inhibit the aggregation of human platelets by acting as agonists at the PGI2 receptor linked to adenylate cyclase. They represent a class of compound with both thromboxane receptor blocking activity and prostacyclin mimetic activity.

Adenylyl Cyclases↗
PubMed1986

Immunoglobulin E-dependent stimulation of human alveolar macrophages: significance in type 1 hypersensitivity.

Human alveolar macrophages were obtained during diagnostic bronchoalveolar lavage. Cells were cultured, and morphological examination (including electron microscopy) revealed that not more than 5% of the cultured cells were identifiable as cells other than alveolar macrophages. The cells were sensitized with human myeloma immunoglobulin E. and then challenged with anti-immunoglobulin E anti-sera. The experiments employed a highly specific monoclonal antibody and three affinity purified reagents. The formation of immunoglobulin E/anti-immunoglobulin E complexes facilitated release from alveolar macrophages of leukotriene B4, prostaglandin F2 alpha, thromboxane B2 and the lysosomal hydrolase N-acetyl-beta-D-glucosaminidase. There was no release of active oxygen species, with this stimulus, as measured by lucigenin chemiluminescence. Immunoglobulin E receptors were identified histochemically on the surface of human alveolar macrophages, and were visualized as conjugates with colloidal gold by electron microscopy. These results support the view that human alveolar macrophages may contribute to type 1 hypersensitivity reactions in the lung.

Asthma↗
PubMed1985

A novel approach for the study of thromboxane A2 and prostaglandin H2 receptors using an 125I-labeled ligand.

Previous studies of eicosanoid receptors have utilized 3H-labeled ligands. However, 125I has a higher theoretical specific activity (approximately 2000 Ci/mmole) than 3H (29 Ci/mmole), which provides a potential advantage for 125I ligand binding studies when the receptor density is low. Since eicosanoids do not possess an easily iodinatable structure (e.g. a phenol or imidazole ring), it is not feasible to directly incorporate 125I into the molecule. The thromboxane A2/prostaglandin H2 receptor antagonist, cis-7-(2-p-hydroxyphenylethanolaminocyclopentyl)-heptanoic acid (cis-APO), was synthesized to test the concept that it could be labeled with 125I and used as a ligand for binding studies. cis-APO is a structural analog of 13-azaprostanoic acid, a TXA2/PGH2 antagonist [G. C. Le Breton, D. L. Venton, S. E. Enke and P. V. Halushka, Proc. natn. Acad. Sci. U.S.A. 76, 4097 (1979)], in which the omega aliphatic chain was substituted with 2-p-hydroxyphenylethanol, which contains a phenolic group. [127I]cis-APO was synthesized by insertion of 127I (stable isotope) into the phenolic portion of the molecule under alkaline conditions. [125I]-cis-APO was synthesized via insertion of 125I (unstable isotope) into the molecule in the presence of chloramine T. cis-APO inhibited human platelet aggregation induced by the thromboxane mimetic U46619 [C. Malmsten, Life Sci. 18, 169 (1976)]. The IC50 for cis-APO was 6.4 +/- 0.7 microM and for [127I]-cis-APO was 9.8 +/- 1.3 microM (P less than 0.001). [125I]-cis-APO binding to a human platelet membrane preparation at 4 degrees was time and protein concentration dependent, saturable, and reduced or abolished by trypsin or boiling respectively. The Kd for iodo-cis-APO determined at equilibrium using a Scatchard analysis was 1.48 microM and the maximum binding capacity was 18.7 pmoles/mg protein. The forward rate constant (k+1) was 2.3 X 10(3) M-1 s-1 and the dissociation constant (k-1) was 2.12 X 10(-3) s-1. The Kd determined from k-1/k+1 was 0.92 microM. These observations show that the omega side chain of eicosanoid analogs can be substituted with a phenolic group, iodinated, and retain biological activity. These molecules may then be utilized to study thromboxane A2 or prostaglandin H2 receptors.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1985

Desensitization of prostacyclin responsiveness in a neuronal hybrid cell line: selective loss of high affinity receptors.

The binding of [3H]-iloprost (ZK36374) to NCB-20 membranes revealed a single population of high affinity receptors (KD = 9.55 nM, Bmax = 431 fmol mg-1 protein) and a low affinity, non-saturable binding component. Desensitization of prostacyclin-responsiveness of NCB-20 cells is induced by culture in the presence of the stable prostacyclin analogue carbacyclin. Desensitization is accompanied by an increase in the Kact value for prostacyclin (64.1 nM to 175 nM), and a reduction in the prostacyclin-dependent increase in adenylate cyclase activity (41.2 to 15.1 pmol cyclic AMP min-1 mg-1 protein). Desensitization is not accompanied by changes in the coupling of the catalytic (C) to the regulatory (Ns) subunit of adenylate cyclase. In addition, the physical identity of the receptor molecule (as characterized by its sensitivity to electron bombardment in the beam of a linear accelerator) is not changed by desensitization. Desensitization of prostacyclin-dependent activation of adenylate cyclase may be explained most simply by a loss of prostacyclin receptors. The anomalous increase in the Kact (concentration of prostaglandin giving half-maximum enzyme activation) for prostacyclin-stimulated adenylate cyclase was not accompanied by a substantial change in the KD of [3H]-iloprost binding, and is explained by a loss of spare receptors. Prostacyclin responsiveness in non-dividing cells may be restored after desensitization by prolonged culture (up to 48 h) in the absence of carbacyclin. Resensitization is accompanied by restoration of the high affinity Kact value (143 nM to 45.5 nM), and is dependent on de novo protein synthesis.

Adenylyl Cyclases↗
PubMed1984

Identification of the prostacyclin receptor by radiation inactivation.

Evidence has been obtained for a specific protein receptor for prostacyclin on cells of the NCB-20 somatic hybrid. A new stable prostacyclin analog, 5-[(E)-(1S,5S,6R,7R) - 7 - hydroxy-6-[(E) - (3S,4RS) -3-hydroxy-4-methyl-1 -octen-6-inyl]bicyclo[3.3.0]-octan-3-ylidene]pentanoic acid (Iloprost, ZK36374) activates adenylate cyclase of NCB-20 cell membranes to an extent similar to prostacyclin and with a comparable high affinity. The binding of [3H]Iloprost to NCB-20 membranes was rapid with an association rate constant (k+1) of 2.01 X 10(5) M-1 s-1 at 20 degrees C. The rate constant for the dissociation of the ligand-receptor complex (k-1) was 1.19 X 10(-3) s-1, giving a dissociation constant (k-1/k+1) of 5.9 nM. The equilibrium dissociation constant was 29.9 nM, and the membranes had a maximum binding capacity of 347 fmol mg-1 protein. Radiation inactivation has been employed to determine the molecular weights of the functional prostacyclin receptor and components of the adenylate cyclase system in the plasma membrane of the NCB-20 cells. Cell membranes were lyophilized prior to irradiation, which lead to the formation of high-molecular-weight aggregates. The aggregation was avoided, however, when membranes were prepared in an isotonic Tris-HCl buffer containing sucrose. Molecular weight values of 111,000 for the catalytic subunit of adenylate cyclase, 89,000 for the regulatory subunit, and 83,000 for the prostacyclin receptor were obtained. Loss of [3H]Iloprost binding capacity after irradiation of lyophilized membranes yielded a molecular weight value (mean +/- S.E.) for the prostacyclin receptor of 82,800 +/- 12,900 (n = 3).

Adenylyl Cyclases↗
PubMed1984

Synthesis of leukotriene B4, and prostanoids by human alveolar macrophages: analysis by gas chromatography/mass spectrometry.

Human alveolar macrophages, obtained during diagnostic bronchoscopy, were maintained in monolayer culture. Challenge of these cells (greater than 95% purity) with 1.2 mg/ml zymosan A particles (opsonized with human serum) was followed by a rapid release of leukotriene B4 into the medium, 7.28 +/- 5.99 ng/mg cell protein at 2 h (mean +/- S.D.4, n = 4). Leukotriene B4 was identified and measured by a novel technique employing capillary column gas chromatography coupled to negative ion chemical ionization mass spectrometry. The release of thromboxane B2, prostaglandins D2, E2, F2 alpha and the lysosomal enzyme N-acetyl-beta-D-glucosaminidase was also measured. Thromboxane B2 was the most abundant metabolite of arachidonic acid released into the culture medium (65.2 +/- 14.8 ng/mg cell protein 2 h after the addition of zymosan A, n = 4), and the synthesis of thromboxane B2 was inhibited by greater than 90% in 1 microM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B4 synthesis.

Gas Chromatography-Mass Spectrometry↗
PubMed1984

Dexamethasone inhibits the production of thromboxane B2 and leukotriene B4 by human alveolar and peritoneal macrophages in culture.

Cultured human alveolar and peritoneal macrophages have been shown to release thromboxane B2 and leukotriene B4. The release was facilitated by stimulation of the macrophages with opsonized zymosan A (1.2 mg/ml). The release was inhibited in a concentration-dependent manner by incubation of the cells with dexamethasone (1 nmol/l to 1 mumol/l).

Acetylglucosaminidase↗
PubMed1983

Neuropeptide Y in neuroblastoma X glioma hybrid cells. Response to dexamethasone and nerve growth factor.

High concentrations of a newly-identified biologically potent peptide, neuropeptide Y, have been demonstrated in 3 related mouse neuroblastoma-derived clonal cell lines, N18TG2 0.35 pmol/mg protein, NG108-15 0.44 pmol/mg protein and NCB-20 0.39 pmol/mg protein. The NG108-15 cell line was chosen for further evaluation. Dexamethasone (10 microM) and nerve growth factor (10 ng/ml) resulted in a 2-fold increase in cellular neuropeptide Y concentrations. The response to dexamethasone was demonstrated to be dose-dependent. Exposure to both agents in combination resulted in a more than additive effect, indicating synergism.

Animals↗
PubMed1983

Epoprostenol (prostacyclin, PGI2) binding and activation of adenylate cyclase in platelets of diabetic and control subjects.

1 The binding of epoprostenol (prostacyclin, PGI2) to isolated fractured human platelets has been studied using tritiated PGI2. 2 High and low affinity binding sites for PGI2 have been identified (Kd values = 16 and 382 nM). 3 Analysis of the prostacyclin-dependent activation of adenylate cyclase suggests that enzyme activation is mediated by the high affinity binding site. 4 Platelet PGI2 receptor binding and adenylate cyclase activation by PGI2 are unchanged in diabetic and normal human platelets. 5 This work suggests that hyperaggregability of diabetic platelets is not due to any alteration of platelet prostacyclin receptor numbers or their activation.

Adenylyl Cyclases↗