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Biomedical subjects

R M Dawson

Publications and source records attributed to R M Dawson.

At least 37 records · Page 2Linked to original sources
PubMed1987

Microencapsulation of CHO cells in a hydroxyethyl methacrylate-methyl methacrylate copolymer.

Chinese hamster ovary fibroblasts, as model cells, have been microencapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer (HEMA-MMA) by interfacial precipitation. The polymer containing approximately equal to 75 mol% HEMA, dissolved in polyethylene glycol 200 (PEG 200) was coextruded with the cell suspension (4-6 X 10(5) cells/ml in the alpha-MEM with 10% foetal calf serum +/- Ficoll 400/PBS) through a concentric needle assembly. Polymer solution droplets, containing cells, were blown off the end of the needle assembly by a coaxial filtered air stream into a nonsolvent bath containing phosphate buffered saline (PBS) with 5 ppm Pluronic L101, overlaid with hexadecane. The nascent capsules hang at the hexadecane/PBS interface while the solvent is extracted into the aqueous nonsolvent, to precipitate the polymer around the cells. The resultant capsules were 500 microns-1 mm in diam. with a microporous sponge-like interior, and also very tough and flexible. The cells survived encapsulation based on subculture ability, retention of some fluorescein diacetate (FDA) activity over 5 d and direct light microscopic evidence of cell growth over 10 d after histological sectioning and staining. However, cell growth was not uniformly observed (especially in the FDA assay) and this was attributed to space limitations for growth within the microporous interior. Continued development of this process and adaptation to cells such as pancreatic islets is expected to lead to hybrid artificial organs which are capable of ameliorating metabolic disorders such as diabetes.

Acrylates↗
PubMed1986

Methodology for benzodiazepine receptor binding assays at physiological temperature. Rapid change in equilibrium with falling temperature.

Benzodiazepine receptors of rat cerebellum were assayed with [3H]-labeled flunitrazepam at 37 degrees C, and assays were terminated by filtration in a cold room according to one of three protocols: keeping each sample at 37 degrees C until ready for filtration, taking the batch of samples (30) into the cold room and filtering sequentially in the order 1-30, and taking the batch of 30 samples into the cold room and filtering sequentially in the order 30-1. the results for each protocol were substantially different from each other, indicating that rapid disruption of equilibrium occurred as the samples cooled in the cold room while waiting to be filtered. Positive or negative cooperativity of binding was apparent, and misleading effects of gamma-aminobutyric acid on the affinity of diazepam were observed, unless each sample was kept at 37 degrees C until just prior to filtration.

Animals↗
PubMed1986

Modification of the effects of muscarinic agonists by reversible and irreversible anticholinesterase compounds in the guinea pig atrium.

The cholinergic agonists acetylcholine (ACh), carbamylcholine and methacholine were found to be equieffective in reducing the force of left atrial contraction, but to differ in their ability to shorten the action potential duration. The irreversible cholinesterase inhibitor soman had no effect on the actions of the non-hydrolyzable agonist carbamylcholine, but potentiated the actions of ACh. The reversible inhibitor edrophonium both potentiated and antagonized the effects of ACh. It antagonized the effects of carbamylcholine and after atrial cholinesterase was inhibited with soman it also antagonized the effects of ACh. Its anticholinesterase action and inhibitory action at the muscarinic receptor were confirmed in separate studies. Edrophonium is approximately 12 times more potent as an anticholinesterase than it is in blocking the muscarinic receptor. However, some actions of edrophonium cannot be explained in the context of its anticholinergic and antiesterase actions. Thus it increases the force of atrial contraction and antagonizes the negative inotropy due to soman. An inhibitory effect on an outward K+ current may be involved. The difference in the ability of the three cholinergic agonists to shorten the action potential may also be related to differences in efficacy at this K+ channel.

Acetylcholine↗
PubMed1986

Methylation of critical carboxyl groups in the vicinity of the sodium channel of guinea-pig atrium.

Methylation of a critical carboxyl group in guinea-pig left atrium with trimethoxonium ion leads to loss of excitability. The critical group(s) could be protected with a number of cationic drugs, so that on washout of the protecting drug and reaction products full excitability returned. Tetrodotoxin, edrophonium, cholinergic agonists and amantadine protected the preparation. During the recovery period readmission of these drugs led to the same pharmacological response as during the control period, suggesting protection of specific site(s). This thesis was confirmed in crossover experiments, in which the atrium was exposed to the methylating agent in the presence of one protecting drug, then exposed to another during recovery. The expected pharmacological responses were obtained. Assay of muscarinic receptors with [3H]-quinuclidinyl benzilate after methylation without protection or in the presence of tetrodotoxin or acetylcholine suggested that these receptors are not involved in the maintenance of excitability. It is postulated that the Na+ channel and the K+ channel are located in the same macromolecular membrane complex, and that the K+ channel has a cholinoreceptive sub-site.

Acetylcholine↗
PubMed1985

Characterization of membrane lipids of a general fatty acid auxotrophic bacterium by electron spin resonance spectroscopy and differential scanning calorimetry.

Lipids in the plasma membrane of the general fatty acid auxotroph Butyrivibrio S2 pack as a bilayer that is characterized by a high order and high motional anisotropy and a low membrane fluidity compared to mammalian plasma membranes. Lipid packing as determined by the electron spin resonance (ESR) order parameter and membrane fluidity as measured by ESR correlation times are, however, comparable to those of other bacterial membranes. Membranes of the organism grown with saturated fatty acids of well-defined hydrocarbon chain length undergo a broad reversible endothermic phase transition, the peak temperature of which is well below the growth temperature; the end-point temperature of this thermal transition approximately coincides with the minimum temperature supporting significant growth of the organism. The lipid phase transition is also reflected in the temperature dependence of various ESR parameters, whereby the transition temperature thus derived is higher than the peak temperature of the endothermic transition but still lower than the growth temperature. ESR and calorimetry evidence taken together suggest that the endothermic transition is a gel to liquid-crystal transition and that, at the growth temperature, the plasma membrane of Butyrivibrio S2 is in the liquid-crystalline state. Similar values were measured for the order parameter of cell membranes of Butyrivibrio S2 regardless of whether the organism was grown on myristic, palmitic, or stearic acid. Butyrivibrio S2 has a mechanism enabling it to maintain membrane packing and fluidity at a fairly constant level.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1985

The inhibition of diacylglycerol-stimulated intracellular phospholipases by phospholipids with a phosphocholine-containing polar group. A possible physiological role for sphingomyelin.

Phosphatidylinositol phosphodiesterase activated by diacylglycerol is substantially inhibited by all phospholipids containing a phosphocholine head group, including phosphatidylcholine, hydrogenated phosphatidylcholine, choline plasmalogen, lysophosphatidylcholine, lysocholine plasmalogen, sphingomyelin and sphingosylphosphocholine. The sphingosine-containing phospholipids are the most inhibitory. Phosphatidic acid does not inhibit, and phosphatidylethanolamine activates the hydrolysis still further. Sphingomyelin is highly inhibitory to a diacylglycerol-stimulated intestinal mucosal phospholipase A2, or a liver lysosomal phospholipase A1 + A2, both hydrolysing a phosphatidylcholine substrate. Sphingomyelin [20% molar (20 mol of sphingomyelin/80 mol of phosphatidylethanolamine)] activates phosphatidylethanolamine hydrolysis by intestinal mucosal phospholipase A2, and then at higher concentrations (40% molar) substantially inhibits the activity. The results are discussed in relation to possible molecular reorganizations brought about in the hydrated phospholipid substrate complex, and in particular the possible stabilizing role of sphingomyelin in the maintenance of membrane structure, and hence in the modulation of phospholipase activity.

Animals↗
PubMed1985

Interactions of amantadine with the cardiac muscarinic receptor.

The antiviral drug amantadine has anticholinergic effects in the guinea-pig atrium at concentrations greater than 1 X 10(-4)M. It is a competitive inhibitor of [3H]-quinuclidinyl benzilate binding to the muscarinic receptor, but antagonizes the negative inotropic effect of acetylcholine in a non-competitive manner. It increases the duration of the atrial action potential and also increases the force of atrial contraction. These effects are evident at approximately 10 times lower concentrations than the antimuscarinic effects. The increase in contractility can be reversed by propranolol (5 X 10(-7)M) but the increase in action potential duration is potentiated by propranolol. Shortening of the action potential duration by acetylcholine was reversed by amantadine, but at approximately ten times lower levels than were needed to reduce the negative inotropic effect. Interactions between beta adrenoceptor binding of [3H]-dihydroalprenolol and amantadine could not be demonstrated. Similarly, binding of [3H]-nitrendipine to the calcium channel is not influenced. It is suggested that amantadine may exert its positive inotropic effect by interaction with the potassium channel, causing a delay in outward current.

Acetylcholine↗
PubMed1984

Long-chain unsaturated diacylglycerols cause a perturbation in the structure of phospholipid bilayers rendering them susceptible to phospholipase attack.

Intracellular phospholipases (A2 and C types) can hydrolyse bilayer-forming phospholipids much more rapidly when diacylglycerol is added. Unsaturated long-chain diacylglycerols are much more effective than short-chain saturated diacylglycerols or 1-oleoyl,2-acetylglycerol. Diacylglycerol does not change the electrokinetic properties of the phospholipid water interface, nor does it enhance enzymic digestion of monolayers. 31P-NMR of phosphatidylcholine indicates that diacylglycerol causes an isotropic component to develop in the spectrum of the bilayers which correlates approximately with the enhancement of phospholipase A2 attack. Addition of further diacylglycerol converts this transitional stage of unknown structural origin to the hexagonal II phase with a total loss of enzyme activity.

Animals↗
PubMed1984

Phosphatidylinositol-4,5-bisphosphate phosphodiesterase and phosphomonoesterase activities of rat brain. Some properties and possible control mechanisms.

The phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] [and to a lesser extent, the phosphatidylinositol-4-phosphate (PtdIns4P)] phosphodiesterase and monoesterase activities of a rat brain supernatant have been studied by using 32P-labelled substrates prepared from human red blood cells. PtdIns(4,5)P2 monoesterase is maximally stimulated by Mg2+, though some activity is detectable in Ca2+/EDTA (Mg2+-free) buffers. The phosphodiesterase, however, is Ca2+-dependent, and in Ca2+/EDTA buffers with the pure lipid as substrate, shows maximal activity at 100 nM-Ca2+. If PtdIns(4,5)P2 is presented as a component of a lipid mixture of similar composition to that of the inner half of the lipid bilayer of a rat liver plasma membrane, the phosphodiesterase shows considerable activity at 1 microM-Ca2+, and is maximal at 100 microM-Ca2+. However, if it is assayed against the same substrate in Ca2+/EGTA buffers with 3mM-Mg2+ and 80 mM-KCl present (as an approximate parallel with the ionic environment in vivo), it shows no detectable activity below 100 microM-Ca2+, and is maximal at 1 mM-Ca2+. The monoesterase can hydrolyse PtdIns(4,5)P2 in such a lipid mixture at all Ca2+ concentrations with 1 or 3 mM-Mg2+ present. PtdIns(4,5)P2 phosphodiesterase can be induced to attack its substrate under ionic conditions similar to those in vivo (0.1-1 microM-Ca2+; 1 mM-Mg2+; 80 mM-KCl) by the conversion of its substrate into a non-bilayer configuration. If given such a substrate [by mixing PtdIns(4,5)P2 with an excess of phosphatidylethanolamine (PtdEtn)] it shows a shallow Ca2+-dependency curve from 0.1 to 100 microM and then a steep rise to 1 mM-Ca2+. Together these observations lead us to the suggestion that a perturbation in a membrane in vivo equivalent to a non-bilayer configuration would be sufficient to induce phosphodiesterase-catalysed PtdIns(4,5)P2 breakdown. When given substrates mixed with excess PtdEtn at pH 7.25 (or 5.5), 1 microM-Ca2+, 1 mM-Mg2+ and 80 mM-KCl, the rat brain supernatant phosphodiesterase activity hydrolysed PtdIns(4,5)P 50-100-fold faster than it hydrolysed phosphatidylinositol (PtdIns). If the supernatant was presented with such a non-bilayer mixture containing a ten-fold excess of PtdIns over PtdIns(4,5)P2, the latter phospholipid was still hydrolysed by phosphodiesterasic cleavage at nearly ten times the rate of the former. Receptor-stimulated phosphodiesterase cleavage of polyphosphoinositides is an early event in cell activation by many agonists. The properties of PtdIns(4,5)P2 phosphodiesterase in vitro suggest that a change in the presentation of its substrate would be a sensitive and sufficient control on the enzyme's activity in vivo.

Animals↗
PubMed1984

The affinity of [3H]quinuclidinyl benzilate and some other radioligands for glass microfiber and cellulose filters in some common liquid scintillation solvents.

The optimum conditions for measuring radioactivity in the filtration assay of muscarinic cholinoceptors with tritiated quinuclidinyl benzilate are to use Whatman GF/B filters and to add a simple toluene scintillant to them while they are still damp. Practically all the radioactive material is then slowly extracted into the scintillant and high counting efficiencies are obtained after 24 h. Dried filters, or dry filters in control experiments in the absence of receptor, adsorb much of the radioactivity with a 30% reduction in counting efficiency. Other scintillants were able to extract the radioactive material from dry filters, but were generally not preferable to toluene. The GF/B filters performed better than other glass microfiber and cellulose filters in terms of retention of receptor-bound ligand, rapid filtration rates, and low filter blanks. Toluene is unsuitable as a scintillant with GF/B filters for some other radioligands examined.

Animals↗
PubMed1984

One-dimensional thin-layer chromatographic separation of the lipids involved in arachidonic acid metabolism.

A one-dimensional thin-layer chromatographic separation technique is described that separates the phospholipids and neutral lipids that are principally involved in arachidonic acid metabolism from each other, and from arachidonic acid and its eicosanoid derivatives. The technique is useful in studies on changes in arachidonic acid metabolism following cell stimulation. Balance sheets may be drawn up between the loss of radioactivity from phospholipids, and the gain in free fatty acid, eicosanoids, and esterified lipids of the phosphatidylinositol cycle.

Animals↗
PubMed1983

Diacylglycerol potentiates phospholipase attack upon phospholipid bilayers: possible connection with cell stimulation.

Many phospholipases of the A or C types show a very limited ability to hydrolyse phospholipids such as phosphatidylcholine or phosphatidylinositol which adopt a bilayer form when hydrated. The addition of unsaturated 1,2 or 1,3 diacylglycerols to such substances produces a marked stimulation of the attack. In contrast, the hydrolysis of phosphatidylethanolamine, which normally exists in the hexagonal II structure, is not stimulated by diacylglycerol. It is suggested that the liberation of diacylglycerol which often occurs during cell stimulation, may play a part in activating the associated cascade of phospholipid reactions.

Animals↗
PubMed1983

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.

Animals↗
PubMed1983

The alkaline phospholipase A1 of rat liver cytosol.

1. Rat liver cytosol contains a heat-sensitive phospholipase A1 active against phosphatidylethanolamine, 1-acylglycerophosphoethanolamine and, to a very much lesser extent, phosphatidylcholine and phosphatidylinositol. 2. Activity towards a pure phosphatidylethanolamine substrate is invoked by the presence of water-soluble cations that do not precipitate at the pH optimum of the enzyme (9.5). In this activation bivalent cations, e.g. Mg2+, Ca2+, Mn2+, Sr2+ and Ba2+, are effective at much lower concentrations (2.5-5 mM) than univalent cations K+, Na+ and NH4+ (100 mM). 3. In the absence of such cations the enzyme can be activated by cationic amphiphiles containing quaternary nitrogen or by basic proteins. 4. It is concluded that these agents activate the enzyme by reducing the negative zeta potential on the substrate at the high pH optimum (9.5) and allow interaction with the enzyme whose isoelectric point is at 7.15. 5. The activated enzyme is markedly inhibited by mixing the phosphatidylethanolamine substrate with many other phospholipids that exist in cell membranes, e.g. phosphatidylcholine, phosphatidylinositol. On the other hand, both phosphatidylcholine and phosphatidylinositol can be hydrolysed much more readily if they are mixed with an excess of phosphatidylethanolamine. 6. Such results on the inhibition and substrate specificity of the enzyme, coupled with birefringence measurements, allow the tentative conclusion that phospholipid substrates are only attacked when they exist in a hexagonal or non-bilayer structure and not in the bilayer (lamellar) form.

Animals↗
PubMed1983

Activation of sarcoplasmic reticular Ca2+ transport ATPase by phosphorylation of an associated phosphatidylinositol.

Approximately 1 mol phosphatidylinositol phosphate is formed per mol isolated Ca2+ transport ATPase when the enzyme is incubated with ATP/Mg2+. The phosphorylation of this enzyme-associated phosphatidylinositol represents the alkylphosphate formation described earlier. The phosphatidylinositol phosphate has been found in the hydrophobic core of the enzyme. A complex of phosphatidylinositol phosphate with protein can be extracted with acidic chloroform/methanol. The protein behaves like proteolipid during chromatography on Sephadex LH 60 and binds the radioactively labelled phosphatidylinositol phosphate. The phosphorylation of approximately 1 mol phosphatidylinositol per 100,000 g protein correlates with an enhancement of the Ca2+ transport ATPase activity which is due to an approximately 7-fold enhanced affinity for Ca2+ and an approximately 2-fold enhanced maximal turnover.

Animals↗