Whooping cough immunization in France and Britain: discussion paper.
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Brain Diseases↗
Biomedical subjects
E M Ross
Publications and source records attributed to E M Ross.
Phosphatidylcholine-promoted interaction of the catalytic and regulatory proteins of adenylate cyclase.
The catalytic protein of rabbit hepatic adenylate cyclase, after chromatographic separation from the GTP-binding regulatory protein (G/F) (Ross, E. M. (1981) J. Biol. Chem. 256, 1949-1953), is essentially free of endogenous phospholipids. This preparation is active in the presence of Mn2+ and is markedly stimulated by forskolin, but it is stimulated only slightly by the addition of purified G/F plus an activator (GTP gamma S or NaF). The ability of activator-liganded G/F to stimulate the activity of the resolved catalyst is increased up to 8-fold by the addition of either dimyristoylphosphatidylcholine (0.3-1.5 mM optimal concentration) or several other phosphatidylcholines. Phosphatidylcholine stabilizes the catalyst to denaturation but has little effect on its basal activity or on its stimulation by Mn2+ or forskolin. It also had no stimulating effect on the activation of G/F by GTP gamma S. These data are interpreted as showing that phosphatidylcholine promotes or is required for the stimulatory interaction of activator-liganded G/F with the catalytic protein of adenylate cyclase. Lubrol 12A9, Triton X-100, cholate, lysophosphatidylcholine, digitonin, and phosphatidylserine could not substitute for phosphatidylcholine. The detergents inhibited stimulation by liganded G/F even in the presence of phosphatidylcholine. Removal of cholate from a mixture of soluble catalytic protein and phosphatidylcholine by dialysis and sucrose density gradient centrifugation caused the binding of catalytic protein to large unilamellar vesicles. This preparation was further reconstituted with increasing amounts of G/F to yield vesicles with varied G/F:catalyst ratios and similarly varied responses to G/F-mediated activating ligands.
Adenylyl Cyclases↗
ABC of 1 to 7: whooping cough.
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Child, Preschool↗
Radioimmunoassay of digitonin.
We describe a radioimmunoassay for digitonin which utilizes the ability of digotonin to compete with 125I-labeled digoxigenin for binding to anti-digoxin antiserum. As performed using the Gammaflo automated radioimmunoassay system and commercially available reagents, the assay can detect as little as 20 micrograms/ml (15 microM) digitonin. The assay is insensitive to interference by cholesterol or other cell membrane constituents and is useable above and below the critical micelle concentration of digitonin. It should be useful for the monitoring of digitonin concentrations in solubilized biochemical preparations.
Animals↗
Functional reconstitution of beta-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase.
A procedure for the functional reconstitution of beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. beta-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of beta-adrenergic agonists. This stimulation of the activation of G/F was specific for beta-adrenergic agonists and could be specifically blocked by beta-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active beta-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F by receptor was reconstituted.
Adenylyl Cyclases↗
Whooping cough and whooping cough vaccine: the risks and benefits debate.
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Disease Outbreaks↗
Reye's syndrome in children under three years old.
The National Childhood Encephalopathy Study identified 37 cases of Reye's syndrome in children aged between 2 and 36 months in a 3-year period, giving an estimated incidence in Great Britain of 0.7/100 000 children a year. The diagnostic features were neither consistently positive in these cases, nor negative in 11 others later considered not to have Reye's syndrome. The prognosis was poor; the fatality rate was 46%, and 60% of the survivors were handicapped. A surveillance scheme to investigate pathological, clinical, and epidemiological factors in this rare condition is required. This has now been set up in the UK.
Child, Preschool↗
Joubert-Boltshauser syndrome with polydactyly in siblings.
Two siblings are described with clinical features of the Joubert-Boltshauser syndrome. Both had polydactyly and one had fleshy tumours of the tongue. Computed tomography of the brain showed hypoplasia of the cerebellar vermis, associated in one case with a cyst of the fourth ventricle.
Apnea↗
Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes.
The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.
Adenosine Diphosphate Ribose↗
Pertussis immunisation and serious acute neurological illness in children.
The first 1000 cases notified to the National Childhood Encephalopathy Study were analysed. The diagnoses included encephalitis/encephalopathy, prolonged convulsions, infantile spasms, and Reye's syndrome. Eighty-eight of the children had had a recent infectious disease, including 19 with pertussis. Only 35 of the notified children (3.5%) had received pertussis antigen within seven days before becoming ill. Of 1955 control children matched for age, sex, and area of residence, 34 (1.7%) had been immunised with pertussis vaccine within the seven days before the date on which they became of the same age as the corresponding notified child. The relative risk of a notified child having had pertussis immunisation within that time interval was 2.4 (p less than 0.001). Of the 35 notified children, 32 had no previous neurological abnormality. A year later two had died, nine had developmental retardation, and 21 were normal. A significance association was shown between serious neurological illness and pertussis vaccine, though cases were few and most children recovered completely.
Acute Disease↗
Physical separation of the catalytic and regulatory proteins of hepatic adenylate cyclase.
Both the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of rabbit hepatic adenylate cyclase were solubilized in active form in sodium cholate at high ionic strength. Upon precipitation by (NH4)SO4, C was selectively aggregated such that it was largely excluded from Ultrogel AcA 34 when chromatographed in cholate solution at high ionic strength. G/F eluted with KD congruent to 0.5. Thus C can be completely resolved from G/F by this procedure. Rabbit hepatic C prepared in this way resembles the C activity previously described by us in the cyc- mutant of S49 lymphoma cells (Ross, E. M., Howlett, A. C., Ferguson, K. M., and Gilman, A. G. (1978) J. Biol. Chem. 253, 6401-6412). Similarities include 7- to 10-fold stimulation by Mn2+ relative to Mg2+ and a complete lack of sensitivity to fluoride or guanine nucleotides. Reconstitution of hepatic C with hepatic G/F causes restoration of stimulation up to 25-fold by either of these activators in the presence of Mg2+. While C is relatively unstable in cholate under these conditions, especially at temperatures over 4 degrees C, it is stabilized by removal of cholate and by addition of phospholipid. These procedures allow the study of the catalytic protein of adenylate cyclase from a hormone-responsive tissue in the absence of endogenous regulatory protein or membrane lipid and also provide a starting point for the purification of the catalyst.
Adenylyl Cyclases↗
Issue for the '80s: cutting the costs of workers' rehabilitation.
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Cost Control↗
Epilepsy in childhood: findings from the National Child Development Study.
By the age of 11 years 1043 children (6.7%) in an unselected national sample had a history of seizures or other episodes of loss of consciousness; 322 (20.8/1000) had a history of febrile convulsions without other epileptic problems. A clear-cut diagnosis of non-febrile epilepsy was established in 64 children (4.1/1000) by the age of 11 on the basis of confirmatory information supplied by family doctors and paediatricians. A further 39 (2.6/1000) were reported as having epilepsy but did not fulfil the study criteria. The progress of 59 of the 64 children with estabished epilepsy was reviewed again when they were aged 16. Of the 37 educated in normal schools eight (22%) had one or more seizures in their 16th year compared with 13 out of 22 (59%) who received special education. A possible cause for epilepsy was found in 17 of the 64 (27%) children, but for the majority there was no obvious reason.
Adolescent↗
Purification of the regulatory component of adenylate cyclase.
The regulatory component (G/F) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rabbit liver plasma membranes has been purified essentially to homogeneity. The purification was accomplished by three chromatographic procedures in sodium cholate-containing solutions, followed by three steps in Lubrol-containing solutions. The specific activity of G/F was enriched 2000-fold from extracts of membranes to 3-4 mumol x min-1 x mg-1 (reconstituted adenylate cyclase activity). Purified G/F reconstitutes guanine nucleotide-, fluoride-, and hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant of S49 murine lymphoma cells. G/F also recouples hormonal stimulation of the enzyme in the uncoupled variant of S49. Preparations of pure G/F contain three polypeptides with approximate molecular weights of 52,000, 45,000, and 35,000. The active G/F protein behaves as a multisubunit complex of these polypeptides. Treatment of G/F with [32P]NAD+ and cholera toxin covalently labels the molecular weight 52,000 and 45,000 polypeptides with 32P.
Adenylyl Cyclases↗
Biochemical properties of hormone-sensitive adenylate cyclase.
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Adenylyl Cyclases↗
Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors.
beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.
Animals↗
Identification and partial characterization of some components of hormone-stimulated adenylate cyclase.
Adenylate cyclase can be resolved into at least two protein components, neither of which by itself catalyzes the formation of cyclic AMP with Mg-ATP as substrate. Mixture of the two reconstitutes Mg-ATP-dependent, fluoride- and Gpp(NH)p-stimulable activity. One, a heat-labile, N-ethylmaleimide-sensitive protein of molecular weight 190,000 can catalyze cyclic AMP formation with Mn-ATP as substrate, and is therefore proposed to be the catalytic moiety of the adenylate cyclase complex. The other protein (or proteins) is more resistant to heating or N-ethylmaleimide, and is proposed to confer upon the catalyst the ability to ultilize Mg-ATP as substrate. It is also required for the regulation of that activity by guanine nucleotides, hormones, and probably fluoride ion. The catalytic protein is found in a phenotypically adenylate cyclase-deficient (AC-) variant of S49 lymphoma cells. The thermostable regulatory protein can be resolved from the catalyst by heat treatment or N-ethylmaleimide treatment of plasma membranes of wild-type S49 cells, rat or rabbit liver, or avian erythrocytes, and is also found in a phenotypically adenylate cyclase-deficient hepatoma cell line. Mixture of AC- S49 membranes, which contain the beta-adrenergic receptor, with a crude detergent-solubilized preparation of the regulatory protein reconstitutes hormone-stimulable adenylate cyclase activity. Binding of the regulatory protein to the membranes is a time- and temperature-dependent process that requires an activating ligand of the adenylate cyclase system [fluoride, Gpp(NH)p].
Adenylyl Cyclases↗
National Childhood Encephalopathy Study: an interim report.
Data from the first year of the National Childhood Encephalopathy Study were reviewed to see whether any relation was apparent between pertussis vaccination and brain disease. Three hundred and eighty-seven cases of encephalitis and other specified neurological conditions in which the children were admitted to hospital were reported, of which 267 satisfied the study criteria. Control children were matched for age with the index cases, and medical and immunisation histories were reviewed. Few of the index cases had been vaccinated within 28 days before admission to hospital, so that no close association between vaccination and brain disease existed in most cases. The number of children who had recently been immunised was too small for any statistically useful conclusion to be reached about the risk associated with pertussis vaccine. The study is continuing.
Brain Diseases↗