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Biomedical subjects

S E Pedersen

Publications and source records attributed to S E Pedersen.

At least 37 records · Page 2Linked to original sources
PubMed1995

Site-selective photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor by azide derivatives of ethidium bromide.

Three azido derivatives of ethidium bromide, a potent noncompetitive antagonist of the nicotinic acetylcholine receptor from Torpedo californica, were synthesized, namely 8-azido-ethidium chloride, 3-azido-ethidium chloride, and 3,8-diazido-ethidium chloride. These derivatives were tested for their ability to interact with the noncompetitive antagonist binding site and the acetylcholine binding sites on the acetylcholine receptor. The derivatives bound to the noncompetitive antagonist site with 2-5-fold lower affinity than did ethidium bromide, as determined by competitive inhibition of [3H]phencyclidine binding, indicating a moderate effect of the azide groups upon binding. Inhibition of [3H]-acetylcholine binding by ethidium and its azide derivatives indicated differential binding to the two agonist sites, with high affinity binding to the same site that exhibits high affinity for d-tubocurarine. Photoaffinity labeling by these derivatives revealed reaction with the alpha and gamma subunits that was specific for the acetylcholine binding sites. Inhibition of labeling by d-tubocurarine showed reaction with alpha subunits at both of the acetylcholine binding sites, whereas reaction with the gamma subunit was consistent with reaction only at the site with high affinity for d-tubocurarine. There was no corresponding reaction with the delta subunit, which forms part of the second acetylcholine binding site, despite reaction with the apposing alpha subunit. The azides, therefore, display preferential reaction with the gamma subunit. The selectivity of the reaction must reflect structural differences between the two sites, and subsequent determination of the labeled site(s) should reveal the nature of the differences.

Acetylcholine↗
PubMed1994

[Budesonide administered via Turbuhaler and a nebulator].

Two hundred and forty-one children with perennial asthma had their normal dose of budesonide (administered via Nebuhaler) reduced by 50%. Asthma control deteriorated in 126 patients to such an extent that budesonide had to be increased to the normal dose. During a subsequent two week run-in these 126 patients were treated with their normal dose of budesonide via Nebuhaler. Thereafter 64 were randomized to treatment with their normal budesonide therapy and 62 to treatment with half their normal budesonide dose via Turbuhaler for nine weeks. Asthma symptoms, peak-flow measurements, and beta 2-agonist use were recorded. Pulmonary function tests, exercise tests and 24 hour urine sample collections were performed. There were no differences between the groups in any of the parameters studied during run-in or during the study period, except for use of beta 2-agonist, which was significantly lower in the Turbuhaler-treated group. We conclude that Turbuhaler is more effective than Nebuhaler in the treatment of asthma. Therefore the dose of budesonide should be reduced when patients are switched from Nebuhaler to Turbuhaler treatment.

Administration, Inhalation↗
PubMed1994

Natural history of carcinoma in situ of the urinary bladder.

Thirty-one patients with primary or secondary carcinoma in situ of the urinary bladder were followed in an intensive surveillance program to study the untreated natural history of carcinoma in situ. Few patients received any treatment for carcinoma in situ. The patients were followed for up to 14 years, to disease progression or decrease from other causes than untreated cancer. All patients except three experienced new tumour growths, of these 50% within the first year of follow-up. At 5 years of follow-up half of the patients had experienced progressing disease, i.e. muscle invasion, upper tract invasive cancer or metastases. Most patients would seem eventually to experience progressing disease. Short-term results of treatment with BCG should be viewed on background of such long-term follow-up of untreated carcinoma in situ.

Aged↗
PubMed1992

Structure of the noncompetitive antagonist-binding site of the Torpedo nicotinic acetylcholine receptor. [3H]meproadifen mustard reacts selectively with alpha-subunit Glu-262.

[3H]Meproadifen mustard, an affinity label for the noncompetitive antagonist site of the nicotinic acetylcholine receptor (AChR), specifically alkylates the AChR alpha-subunit when the acetylcholine-binding sites are occupied by agonist (Dreyer, E. B., Hasan, F., Cohen, S. G., and Cohen, J. B. (1986) J. Biol. Chem. 261, 13727-13734). In this report, we identify the site of alkylation within the alpha-subunit as Glu-262. AChR-rich membranes from Torpedo californica electric organ were reacted with [3H]meproadifen mustard in the presence of carbamylcholine and in the absence or presence of nonradioactive meproadifen to define specific alkylation of the noncompetitive antagonist site. Alkylated alpha-subunits were isolated and subjected to chemical or enzymatic cleavage. When digests with CNBr in 70% trifluoroacetic acid or 70% formic acid were fractionated by gel filtration high performance liquid chromatography (HPLC), specifically labeled material was recovered in the void volume fractions. Based upon NH2-terminal sequence analysis, for both digests, the void volume fractions contained a fragment beginning at Gln-208 before the M1 hydrophobic sequence, whereas the sample from the digest in trifluoroacetic acid also contained as a primary sequence a fragment beginning at Thr-244 and extending through the M2 hydrophobic sequence. Sequence analysis revealed no release of 3H for the sample from digestion in formic acid, whereas for the trifluoroacetic acid digest, there was specific release of 3H in cycle 19, which would correspond to Glu-262. This site of alkylation was confirmed by isolation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase HPLC of a specifically labeled fragment from an endoproteinase Lys-C digest of the alkylated alpha-subunit. NH2-terminal amino acid sequencing revealed release of 3H at cycle 20 from a fragment beginning at Met-243 and extending into the M3 hydrophobic sequence. Because [3H]meproadifen mustard contains, as its reactive group, a positively charged quaternary aziridinium ion, Glu-262 of the alpha-subunit is identified as a contributor to the cation-binding domain of the noncompetitive antagonist-binding site and thus of the ion channel.

Affinity Labels↗
PubMed1992

[Dietary treatment of diabetes mellitus. Background and rationale for recommendations in the 1990's].

Individual dietary regulation is still an important part of all forms of treatment of diabetes. In insulin dependent diabetes (IDDM) it is rational to advise the patient 1) to arrange his diet so that this results in a low glycaemic response, which implies a relatively high intake of dietary fibre and polysaccharides, 2) to distribute the food into 5-6 daily meals and 3) to consume a low-fat diet. This prevents too pronounced postprandial hyperglycaemia and hypoglycaemia between meals. Simultaneously, insulin sensitivity is increased and not only the insulin requirement but also peripheral hyperinsulinism tend to be reduced. Dietary regulation in IDDM is thus a compensation for the defective synchronization of variations in the plasma levels of glucose and insulin in the present day forms of insulin therapy. Nine out of ten diabetic patients are non-insulin dependent (NIDDM). The great majority are obese, 50% have essential hypertension and just as many have dyslipidaemia (raised serum triglyceride and reduced serum high density lipoprotein (HDL)-cholesterol). The condition is characterized pathophysiologically by insulin resistance in muscle, fat and liver tissue and delayed and frequently reduced glucose-stimulated secretion of insulin. The most important element in dietary regulation in NIDDM is, therefore, reduction of the energy content of the food with the object of achieving and maintaining reduction in weight. Even moderate reduction, in the majority of NIDDM patients, will have the effect that metabolism of carbohydrates and lipids becomes approximately normal on account of considerable increase in insulin sensitivity and to a lesser degree increased secretion of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Glucose↗
PubMed1990

Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor alpha-subunit by epitope mapping.

Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the alpha-subunit between alpha-156 and alpha-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing alpha-subunit were distinguished in terms of their ability to bind alpha-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10 kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease alpha-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both anti-V8-18 and anti-V8-20 mAbs was determined to be within alpha-156 to alpha-179 by isolation of small immunoreactive peptides from proteolytic digests of the alpha-subunit, while the mAb reactive to V8-10 was bound to an epitope within alpha-339 to alpha-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to alpha-147 to alpha-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease alpha-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of alpha-156 and alpha-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.

Amino Acid Sequence↗
PubMed1990

d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor.

The competitive nicotinic antagonist d-[3H]tubocurarine was used as a photoaffinity label for the acetylcholine binding sites on the nicotinic acetylcholine receptor (AcChoR) from Torpedo. Irradiation with 254-nm UV light of AcChoR-rich membranes equilibrated with d-[3H]tubocurarine resulted in covalent incorporation into the alpha, gamma, and delta subunits that could be blocked by alpha-bungarotoxin or by carbamoylcholine. The concentrations of d-[3H]tubocurarine required for half-maximal specific incorporation into the gamma and delta subunits were 40 nM and 0.9 microM, respectively, consistent with the dissociation constants for the high- and low-affinity binding sites (Kd = 35 nM and 1.2 microM). The concentration dependence of incorporation into alpha subunit was biphasic and consistent with labeling of both the high- and low-affinity d-tubocurarine binding sites. The specific photolabeling of each AcChoR subunit was inhibited by carbamoylcholine with appropriate dose dependence. These results establish that, in addition to the alpha subunits, the gamma and delta subunits also contribute directly to the acetylcholine binding sites and that each binding site is at an interface of subunits. Because the AcChoR subunits are homologous and are arranged pseudosymmetrically about a central axis, the photolabeling results are inconsistent with an arrangement of subunits in the AcChoR rosette of alpha beta alpha gamma delta and indicate that either the gamma or delta subunit resides between the alpha subunits.

Animals↗
PubMed1987

Visualization of the cytoplasmic surface of Torpedo postsynaptic membranes by freeze-etch and immunoelectron microscopy.

The synapse-specific Mr 43,000 protein (43K protein) and the acetylcholine receptor were visualized by freeze-etch immunoelectron microscopy in preparations of purified Torpedo postsynaptic membranes. Vesicles were immobilized on glass and then sheared open by sonication to expose the cytoplasmic surface. Membranes were labeled with monoclonal antibodies to the 43K protein or the acetylcholine receptor. The cytoplasmic surface was devoid of filamentous structure, and the 43K protein and the cytoplasmic projection of the acetylcholine receptor were associated with prominent surface particles. Acetylcholine receptor and 43K protein, in membrane surfaces in direct contact with glass coated with polyornithine, segregated into dense particle aggregates separated by smooth membrane patches, whereas those in contact with glass coated with Alcian Blue underwent little or no detectable rearrangement. After treatment of vesicles at alkaline pH to remove the 43K protein, the cytoplasmic surfaces were still covered by a dense array of particles that were more uniform in shape and appeared slightly shorter than those seen on unextracted membranes, but similar in height to the extracellular projection. Monoclonal antibodies to the acetylcholine receptor labeled these particles, while antibodies to 43K protein did not. We conclude that the 43K protein is in direct association with the receptor and that complexes of the receptor and 43K protein can undergo surface-induced lateral redistribution. In addition, the cytoplasmic projection of the acetylcholine receptor is sufficiently large to be readily detected by freeze-etch electron microscopy and is similar in height to the extracellular projection.

Animals↗
PubMed1986

Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit.

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.

Acetylglucosaminidase↗
PubMed1985

Functional activation of beta-adrenergic receptors by thiols in the presence or absence of agonists.

Treatment of beta-adrenergic receptor with dithiothreitol (DTT) or other thiol compounds caused its functional activation in the presence or absence of agonist ligands. Such activation was observed in reconstituted unilamellar phospholipid vesicles that contained beta-adrenergic receptors, purified to greater than or equal to 95% homogeneity from turkey erythrocyte plasma membranes, and the stimulatory GTP-binding protein of the adenylate cyclase system (Gs) purified from rabbit liver. Incubation of the vesicles with 2-10 mM DTT at 0 degrees C for 1 h increased the rate (4-5-fold) and the extent (3-4-fold) of activation of Gs by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding, an effect about equivalent to the addition of beta-adrenergic agonists. Treatment with DTT also markedly potentiated the ability of agonists to stimulate GTP gamma S binding, increasing the initial rate about 10-fold. DTT treatment was as effective as agonist in stimulating GTPase activity, and maximal stimulation was obtained when DTT-treated vesicles were assayed in the presence of agonist. Other thiol compounds produced effects similar to those of DTT but were at least 10-fold less potent. Stimulation of GTP gamma S binding or GTPase activity required active receptor, and treatment of the receptor with DTT prior to reconstitution also increased its efficacy. There was no effect of DTT on Gs alone. Thus, the site of action of DTT appears to be on the beta-adrenergic receptor itself, and the reduction of disulfides and the binding of agonist act synergistically to activate the receptor. DTT treatment made the receptor more labile to thermal denaturation. Inclusion of cholesterol or cholesteryl-hemisuccinate (5-25%) in the vesicles protected the reduced receptor against such denaturation and enhanced its recovery during reconstitution. No effect of cholesterol or cholesteryl-hemisuccinate was observed on the stability of the nonreduced receptor, which was comparable to that observed in native membranes.

Adenylyl Cyclases↗
PubMed1984

Reconstitution of catecholamine-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase.

The stimulatory GTP-binding protein (Gs) of adenylate cyclase, purified from rabbit liver, and beta-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of Gs to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) binding to Gs and [125I]iodocyanopindolol binding to receptors. Activation of reconstituted Gs by GTP gamma S was stimulated up to 10-fold by the addition of the beta-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [35S]GTP gamma S could also be measured in the vesicles and was shown to parallel the functional activation of Gs under all conditions. Most of the assayable Gs in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]GTP gamma S binding were complete in less than 3 min, even under suboptimal conditions, and could go to completion in less than 20 s under maximal stimulation. Agonist-stimulated binding did not require appreciable free Mg2+ (less than 0.1 mM). Activation in the absence of agonist was stimulated by free Mg2+, but maximal activation took up to 10 min in the presence of 50 mM MgCl2. Reconstitution increased the stability of Gs to thermal denaturation. The addition of beta-adrenergic agonist further stabilized Gs, presumably by the formation of a stable agonist-receptor-Gs complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity.

beta-Adrenergic receptors were partially purified from turkey erythrocyte membranes by alprenolol-agarose chromatography to 0.25-2 nmol/mg of protein, and the stimulatory guanosine 5'-triphosphate (GTP) binding protein of adenylate cyclase (Gs) was purified from rabbit liver. These proteins were reconstituted into phospholipid vesicles by addition of phospholipids and removal of detergent by gel filtration. This preparation hydrolyzes GTP to guanosine 5'-diphosphate (GDP) plus inorganic phosphate (Pi) in response to beta-adrenergic agonists. The initial rate of isoproterenol-stimulated hydrolysis is approximately 1 mol of GTP hydrolyzed min-1 X mol-1 of Gs. This low rate may be limited by the hormone-stimulated binding of substrate, since it is roughly equal to the rate of binding of the GTP analogue guanosine 5'-O-(3-[35S] thiotriphosphate) [( 35S]GTP gamma S) to Gs in the vesicles. Activity in the absence of agonist, or in the presence of agonist plus a beta-adrenergic antagonist, is 8-25% of the hormone-stimulated activity. Guanosinetriphosphatase (GTPase) is not saturated at 10 microM GTP, and the response to GTP is formally consistent either with the existence of multiple Km's or of a separate stimulatory site for GTP. The GTPase activity of Gs in vesicles is also stimulated by 50 mM MgCl2 in the presence or absence of receptor. Significant GTPase activity is not observed with Lubrol-solubilized Gs, although [35S]-GTP gamma S binding is increased by Lubrol solubilization.

Adenylyl Cyclases↗