Biomedical subjects

D S Lester

Publications and source records attributed to D S Lester.

42 records · Page 3Linked to original sources

PubMed1990

The role of membrane biophysical properties in the regulation of protein kinase C activity.

Under physiological conditions, protein kinase C is active when bound to membranes. Like most membrane-bound enzymes, its activity is dependent on the nature of its lipid environment. In this article, Richard Epand and David Lester describe the relationship between the ability of specific membrane-active agents to alter the biophysical properties of the lipid environment and their potential to modulate protein kinase C activity. They argue that this can lead to a greater understanding of the mechanism of inhibition and activation of protein kinase C through modulation of the bulk biophysical properties of the membrane, and may provide a new approach to the development of a more specific set of inhibitors.

Animals↗

PubMed1990

Identification of protein kinase C and its potential substrate in Entamoeba histolytica.

1. Protein kinase C (PKC) activity has been identified in various strains of the human parasite, Entamoeba histolytica. 2. An amoebic protein of mol. wt 78,000 was recognized by polyclonal antibodies raised against the 82,000 mol. wt rat brain protein kinase C. 3. A partially purified PKC preparation from E. histolytica phosphorylated histone I in the presence of calcium, phospholipids and diacylglycerol, and specifically bound tritiated phorbol ester at an apparent KD of 9 nM. 4. A relocalization of the amoebic PKC activity from the cytosol to the membrane fraction was observed when trophozoites were actively phagocytising bacteria. Under these conditions, a labelled phosphoprotein of mol. wt 68,000 was identified. 5. Similar to what was found during macrophage activation, a myristoylated mol. wt 68,000 protein was detected in amoebae grown in the absence of bacteria, but not in amoebae which were active in phagocytosis.

Animals↗

PubMed1989

High-pressure extraction of membrane-associated protein kinase C from rat brain.

Extraction of rat brain membrane-associated protein kinase C with high specific activity was obtained by applying benzyl alcohol (a membrane fluidizer), EDTA, and high hydrostatic pressures. Approximately 50% of total brain-associated activity was extracted from membranes. The pressure-extracted activity had an eightfold enrichment in the lipid/protein ratio when compared with the cytosolic fraction. This may explain the inability of exogenous diacylglycerol to stimulate endogenous phosphorylation in pressure-extracted activity. The enzyme is extracted at greater than 1,300 atm, a result indicating it most likely has a portion inserted into the hydrophobic portion of the membrane bilayer. Perturbation of the native membrane induces a change in the membrane-associated protein kinase C-lipid interaction that permits extraction under conditions used for the cytosolic species. This is the first report of conversion of the endogenous membrane species to a cytosolic one and may be important in determining the role of protein kinase C in neuronal regulation.

Animals↗

PubMed1988

Characterization of cAMP-induced activation of epithelial sodium channels.

Incubating toad bladder with 10 mU/ml vasopressin increases the amiloride-blockable Na+ flux in membrane vesicles derived from the epithelial cells by about twofold. This stimulation is further enhanced by 3-isobutyl-1-methylxanthine and can be mimicked by 8-bromoadenosine 3', 5'-cyclic monophosphate. Thus the natriferic action of cAMP involves a sustained change of the apical membrane preserved by the isolated vesicles. The possibility that transport is modulated by direct phosphorylation/dephosphorylation of the Na+ channel was tested. Trapping purified cAMP-dependent protein kinase, cAMP, and ATP in apical vesicles failed to alter Na+ transport even though the enzyme proved active and could phosphorylate intravesicular proteins. Trapping several phosphatases partially purified from toad bladder in vesicles was ineffective as well. These data suggest that the cAMP-induced increase in Na+ conductance involves processes other than phosphorylation of the channel protein or direct channel-cAMP interaction.

1-Methyl-3-isobutylxanthine↗

PubMed1986

Developmental changes in choline uptake and acetylcholine metabolism in the larval brain of the tobacco hornworm, Manduca sexta.

The larval brain of the tobacco hornworm, Manduca sexta, was maintained in vitro and the uptake of labelled and unlabelled choline as well as their subsequent metabolism were measured by high-voltage paper electrophoresis. Significant levels of choline lipid metabolites, phosphorylcholine and acetylcholine (ACh) were noted. Unbound choline reached equilibrium after 6-8 h of incubation, while ACh accumulation continued to increase after 24 h indicating that the rate of synthesis exceeded the rate of breakdown. An apparent Km could not be determined for these whole-organ studies; however, the Vmax for ACh accumulation for days 5 (70 pmol/brain/h) and 6 (105 pmol/brain/h) of the last larval instar did vary significantly while the level of unbound choline in the brain did not change. The level of choline uptake was dependent upon the presence of Na+ and Ca2+, while the amount of ACh accumulated was affected specifically by the presence of Mg2+, the latter ion activating acetylcholinesterase. The determination of levels of unbound choline and ACh accumulation in the developing brain during the last two larval instars demonstrated increases in acetylcholine accumulation at previously reported times of the release of the hormone that initiates the molting process, prothoracicotropic hormone. These changes in the patterns of ACh accumulation occur during 4-8 h time intervals; this is the first report of such short-range changes in neurotransmitter metabolism in whole brains. The intensity of the ACh accumulation shift is equivalent to the intensity of the hormone burst. Other fluctuations in the levels of ACh accumulation and free choline correlate with the development of the brain.

Acetylcholine↗

PubMedDate not supplied

Virtual neuropathology: three-dimensional visualization of lesions due to toxic insult.

A first-pass approach incorporating high-field magnetic resonance imaging (MRI) was used for rapid detection of neuropathologic lesions in fixed rat brains. This inherently 3-dimensional and nondestructive technique provides high-resolution, high-contrast images of fixed neuronal tissue in the absence of sectioning or staining. This technique, magnetic resonance microscopy (MRM), was used to identify diverse lesions in 2 well-established rat neurotoxicity models. The intrinsic contrast in the images delineated lesions that were identified using a battery of histologic stains, some of which would not be used in routine screening. Furthermore, the MRM images provided the locations of lesions, which were verified upon subsequent sectioning and staining of the same samples. The inherent contrast generated by water properties is exploited in MRM by choosing suitable pulse sequences, or proton stains. This approach provides the potential for a comprehensive initial MRM screen for neurotoxicity in preclinical models with the capability for extrapolation to clinical analyses using classical MRI.

Animals↗