Search PubMed⌕ Search

Biomedical subjects

E M Ross

Publications and source records attributed to E M Ross.

At least 55 records · Page 3Linked to original sources
PubMed1994

Childhood epilepsies--a measure of concern.

Childhood epilepsies are a serious and sizeable problem, and children with epilepsy have many needs. Epidemiological studies carried out 20-30 years ago provided valuable information on the incidence and prevalence of epilepsy in children. However, the causes of epilepsies in most children still remain unidentified. Following the introduction of an international classification of epilepsies and sophisticated brain scanning techniques, new population-based studies should now be performed to determine the prevalence and long-term outcome of the various types of epilepsy in children. These studies will undoubtedly lead to a greater understanding of childhood epilepsies and should result in better management of this condition.

Adolescent↗
PubMed1993

Outcome of childhood status epilepticus and lengthy febrile convulsions: findings of national cohort study.

OBJECTIVE: To study outcome after lengthy febrile convulsions and status epilepticus in children. DESIGN: Population based birth cohort study. SETTING: The child health and education study (16,004 neonatal survivors born in one week in April 1970). SUBJECTS: Information available for 14,676 children. OUTCOME MEASURES: Clinical information and tests of intellectual performance at five and 10 years after birth. RESULTS: 19 children had lengthy febrile convulsions and 18 had status epilepticus. Two children with status epilepticus died (one at 5 years old); neither death was directly due to the status epilepticus. Four of the 19 (21%) developed afebrile seizures after lengthy febrile convulsions compared with 14 of the 17 (82%) survivors after status epilepticus. Measures of intellectual performance were available for 33 of the 35 survivors: 23 were normal and 10 were not normal but eight of them had preceding developmental delay or neurological abnormality. CONCLUSION: The outcome in children after lengthy febrile convulsions and status epilepticus is better than reported from studies of selected groups and seems determined more by the underlying cause than by the seizures themselves.

Child↗
PubMed1993

GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction.

Acetylcholine↗
PubMed1993

Regulation of the M1 muscarinic receptor-Gq-phospholipase C-beta pathway by nucleotide exchange and GTP hydrolysis.

M1 muscarinic cholinergic receptors, G1 and G11 (Gq/11), and phospholipase C-beta 1 were highly purified from both natural sources and cells that express the appropriate cDNA's. When the proteins were co-reconstituted into phospholipid vesicles, the receptor efficiently and selectively promoted the activation of Gq/11, leading to marked stimulation of PLC activity in the presence of GTP gamma S. No stimulation was observed in the presence of GTP, however, which led to the finding that PLC-beta 1 stimulates the hydrolysis of GQ/11-bound GTP at least 50-fold. Thus, PLC-beta 1 is a GTPase activating protein, a GAP, for its physiologic regulator Gq/11. We discuss the implications of PLC-beta 1's GAP activity on the M1 muscarinic cholinergic signaling pathway.

Animals↗
PubMed1992

Epilepsy in the first 10 years of life: findings of the child health and education study.

OBJECTIVES: To identify children with afebrile seizures in a national cohort, classify the seizures, and document progress in the first 10 years of life. DESIGN: Population based birth cohort study. SETTING: The child health and education study, which includes 16,004 neonatal survivors (98.5% of infants born in the United Kingdom during one week of April 1970). SUBJECTS: 14,676 children for whom relevant information was available. MAIN OUTCOME MEASURES: Responses to parental and general practitioner questionnaires and hospital records at 5 and 10 years after birth. RESULTS: 84 children (42 boys, 42 girls) had had one or more afebrile seizure (incidence 5.7/1000). 63 children (31 boys, 32 girls) had epilepsy (incidence 4.3/1000). 49 of 55 children had a second seizure within a year of the first. The commonest seizure types were tonic-clonic (42) and complex partial (25). A greater proportion of children with complex partial seizures had recurrences. Children who had infantile spasms or a mixed seizure disorder had a poor outcome. All six children who died had symptomatic seizures in the first year, but seizures were not the direct cause of death. CONCLUSIONS: The results of this study are probably representative of seizure patterns in the general population. Outcome after seizures is determined more by the underlying disease than by the seizures themselves.

Age Factors↗
PubMed1992

Phospholipase C-beta 1 is a GTPase-activating protein for Gq/11, its physiologic regulator.

Purified M1 muscarinic cholinergic receptor and Gq/11 were coreconstituted in lipid vesicles. Addition of purified phospholipase C-beta 1 (PLC-beta 1) further stimulated the receptor-promoted steady-state GTPase activity of Gq/11 up to 20-fold. Stimulation depended upon receptor-mediated GTP-GDP exchange. Addition of PLC-beta 1 caused a rapid burst of hydrolysis of Gq/11-bound GTP that was at least 50-fold faster than in its absence. Thus, PLC-beta 1 stimulates hydrolysis of Gq/11-bound GTP and acts as a GTPase-activating protein (GAP) for its physiologic regulator, Gq/11. GTPase-stimulating activity was specific both for PLC-beta 1 and Gq/11. Such GAP activity by an effector coupled to a trimeric G protein can reconcile slow GTP hydrolysis by pure G proteins in vitro with fast physiologic deactivation of G protein-mediated signaling.

Animals↗
PubMed1992

Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1.

We describe the reconstitution using purified proteins of the m1 muscarinic cholinergic pathway that activates phosphatidylinositol 4,5-bisphosphate-specific phospholipase C via the G protein Gq/11. Recombinant m1 muscarinic receptor was co-reconstituted in lipid vesicles with either hepatic Gq/11 or with cerebral alpha q/11 and beta gamma subunits. The rate of [35S]GTP gamma S binding to the reconstituted vesicles was stimulated 20-50-fold by agonist. Maximal receptor-catalyzed binding was 7 mol of GTP gamma S bound per mol of receptor. The m2 muscarinic receptor was a poor activator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesicles indicated that the receptor could maintain up to 40% of the total coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is consistent with the rate predicted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeric G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indicated that the receptor catalyzed binding to both alpha q and alpha 11 with about equal efficiency. Receptor-catalyzed activation of Gq/11 by GTP gamma S, measured as the ability to activate purified phospholipase C-beta 1, paralleled receptor-catalyzed [35S]GTP gamma S binding. Co-reconstitution of receptor, Gq/11, and phospholipase C-beta 1 restored GTP gamma S-dependent carbachol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 receptor, Gq/11, and phospholipase C-beta 1 are thus sufficient to initiate the hormonal inositol trisphosphate/diacylglycerol signaling pathway without additional proteins.

Animals↗
PubMed1992

Relationship between fetal acidemia at cordocentesis and subsequent neurodevelopment.

To determine whether there is a relationship between chronic fetal acidemia and subsequent neurodevelopment, a follow-up study was undertaken of 36 children with normal karyotype and morphology, who had prenatal cordocentesis for severe growth retardation. The main outcome measure was the Griffiths neurodevelopmental quotient. The children who had acidemia as fetuses (n = 13) had a significantly lower developmental quotient (mean = 91.8, SD = 6.3) than those with normal (n = 23) fetal blood pH (mean = 100.3, SD = 10.3; t = -2.68, p = 0.011). There was also a significant correlation between developmental quotient and the degree of fetal acidemia (r = 0.41, n = 36, p = 0.012). The pregnancies with acidemic fetuses had similar epidemiological characteristics to those with fetuses with a normal pH, except for a higher incidence of smoking. There was no significant correlation between the degree of growth retardation (birth weight expressed as multiples of SD from the mean for gestational age and sex) and fetal acidemia (r = -0.23, n = 36, NS) or subsequent Griffiths developmental quotient (r = -0.005, n = 36, NS). The results show an association between chronic fetal acidemia and subsequent impaired neurodevelopment. This observation suggests that future preventative interventions may be possible.

Journal Article↗
PubMed1991

ATP-dependent regulation of flagellar adenylylcyclase in gametes of Chlamydomonas reinhardtii.

Adenylylcyclase activity in the flagella of gametes of Chlamydomonas reinhardtii was inhibited by prior incubation at or below 30 degrees C in the presence of ATP. This decrease did not occur in the absence of ATP, in the presence of the ATP analog 5'-adenylylimidodiphosphate (App(NH)p), or in the presence of ATP plus the protein kinase inhibitor staurosporine (2 microM). If ATP treatment was performed in the absence of an ATP-regenerating system, activity initially declined and subsequently recovered. Incubation of flagella at 45 degrees C in the absence of ATP or incubation at lower temperatures in the presence of either App(NH)p or staurosporine both increased adenylylcyclase activity (over 10-fold) and blocked subsequent ATP-dependent loss of activity at 30 degrees C. This heat-induced activation was prevented by the presence of ATP plus an ATP-regenerating system. Incubation of flagella with [gamma-32P]ATP followed by gel electrophoresis in sodium dodecyl sulfate indicated the presence of endogenous protein kinase and protein phosphatase activities. These data suggest that the flagellar adenylylcyclase in Chlamydomonas gametes is inhibited by phosphorylation and stimulated by dephosphorylation. This mechanism for regulating adenylylcyclase may underlie the rapid increase in cyclic AMP that is induced by flagellar adhesion during fertilization in Chlamydomonas.

Adenosine Triphosphate↗
PubMed1991

Selectivity of the beta-adrenergic receptor among Gs, Gi's, and Go: assay using recombinant alpha subunits in reconstituted phospholipid vesicles.

The selective regulation of Gs (long and short forms), Gi's (1, 2, and 3), and Go by the beta-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein beta gamma subunits, and individual recombinant G-protein alpha subunits that were expressed in and purified from Escherichia coli. Receptor and beta gamma subunits were incorporated into phospholipid vesicles, and the alpha subunits bound to the vesicles stoichiometrically with respect to beta gamma. Efficient regulation of alpha subunit by receptor required the presence of beta gamma. Regulation of G proteins was measured according to the stimulation of the initial rate of GTP gamma S binding, steady-state GTPase activity, and equilibrium GDP/GDP exchange. The assays yielded qualitatively similar results. GDP/GDP exchange was a first-order reaction for each subunit. The rate constant increased linearly with the concentration of agonist-liganded receptor, and the dependence of the rate constant on receptor concentration was a reproducible measurement of the efficiency with which receptor regulated each G protein. Reconstituted alpha s (long or short form) was stimulated by receptor to approximately the extent described previously for natural Gs. Both alpha i,1 and alpha i,3 were regulated with 25-33% of that efficiency. Stimulation of alpha o and alpha i,2 was weak, and stimulation of alpha o was barely detectable over its high basal exchange rate. Reduction of the receptor with dithiothreitol increased the exchange rates for all G proteins but did not alter the relative selectivity of the receptor.

Animals↗
PubMed1991

Mapping of the mastoparan-binding site on G proteins. Cross-linking of [125I-Tyr3,Cys11]mastoparan to Go.

Mastoparan (MP) activates GTP-binding regulatory proteins (G proteins) by promoting GDP/GTP exchange through a mechanism similar to that of G protein-coupled receptors (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186). [Tyr3, Cys11]MP was synthesized and shown to have regulatory activity similar to that of mastoparan when assayed in the presence of dithiothreitol (DTT). Activation by [Tyr3,Cys11]MP in the absence of DTT was complex in its kinetics, concentration dependence, and dependence on detergents. [125I-Tyr3,Cys11]MP bound covalently to the alpha subunit of G proteins. Cross-linking was blocked by mastoparan or [Tyr3,Cys11]MP. Cross-linking was enhanced by the addition of beta gamma subunits, but no cross-linking to beta gamma subunits was observed. Cross-linking was inhibited by incubation of Go with guanosine 5'-O-(thiotriphosphate) and Mg2+ and was reversed by incubation with DTT or 2-mercaptoethanol. Stoichiometry of labeling was consistent with the cross-linking of one molecule of [125I-Tyr3,Cys11]MP/alpha subunit, and CNBr hydrolysis of the [125I-Tyr3,Cys11]MP-alpha o adduct yielded one major labeled peptide fragment of approximately 6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant alpha o showed that cross-linking occurred at Cys3. No alpha o sequence was obtained from the same fragment prepared from bovine brain alpha o, which is blocked by a myristoyl group at Gly2. Regulation of Go by MP was eliminated by tryptic proteolysis of the amino-terminal region. These observations suggest that the amino-terminal region of G protein alpha subunits contributes to the mastoparan-binding site, which may also be the receptor-binding site, and is involved in regulation of nucleotide exchange.

Amino Acids↗
PubMed1991

Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian beta-adrenergic receptor.

A series of mutant avian beta-adrenergic receptors with progressively truncated carboxyl termini have been expressed in insect and mammalian cells. Removal of 18-124 amino acid residues caused multiple phenotypic changes in the receptor. Membranes from cells that expressed the truncated receptors displayed elevated basal (2- to 3-fold) and agonist-stimulated adenylylcyclase activities. Adenylylcyclase activity in these membranes also displayed greater stimulation in response to partial agonists. Activity was also markedly stimulated by beta-adrenergic ligands that are usually considered to be antagonists (alprenolol, greater than 4-fold; propranolol, approximately 2-fold). Wild type receptor did not mediate a response to these classical antagonists. After purification and reconstitution with Gs, the truncated receptors did not appear to be more active than the wild type. Guanine nucleotides modulated the affinity of agonist for the truncated receptors, whereas the affinity of agonist for the wild type receptor was not altered by guanine nucleotides. The truncated receptors were solubilized from the membrane more efficiently and were more susceptible to amino-terminal proteolysis than was the wild type protein. These results suggest interaction of the carboxyl terminus of the avian beta-adrenergic receptor with cellular regulatory or structural elements.

Adenylyl Cyclases↗
PubMed1991

Immunologic mapping of the amino- and carboxy-termini of the turkey erythrocyte beta-adrenergic receptor: selective proteolysis of both domains.

Peptide-directed antibodies were used to map the N- and C-termini of the turkey erythrocyte beta-adrenergic receptor, the full length recombinant receptor expressed in Sf9 cells, and a mutant that terminates after residue 424 (T424). Both forms of the natural receptor (P40 and P50) were proteolytically clipped between residues 419 and 424. P40, but not P50, is also proteolyzed between residues 14 and 28. Truncation mutants, but not full length receptors, also display both large and small forms. The short form of T424 is formed by proteolysis after residue 14, but neither form is proteolyzed in the C-terminal region. The wild type recombinant receptor is not proteolyzed.

Amino Acid Sequence↗
PubMed1991

Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells.

The turkey beta-adrenergic receptor (beta-AR), the m1 and m2 forms of the human muscarinic cholingeric receptor (MAChR) and several other mutant and wild-type G protein-coupled receptors were produced in insect Sf9 cells by infection with recombinant baculoviruses. Maximal expression for most receptors was 5-30 pmol receptor/mg protein (2-15 nmol/liter culture). The receptors displayed typical ligand binding characteristics. The beta-AR was glycosylated; electrophoretic behavior of the two MAChRs also suggested glycosylation. The beta-AR stimulated endogenous adenylyl cyclase in response to beta-adrenergic agonists. The beta-AR and both MAChRs were purified and coreconstituted with various purified G proteins in phospholipid vesicles. The recombinant beta-AR catalyzed the agonist-dependent activation of Gs by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with the same efficiency as did the natural beta-AR. The m2 MAChR efficiently catalyzed GTP gamma S binding to Go and to the recently identified G protein Gz (Gx). The m2 MAChR also catalyzed the activation of Gj,1 and Gj,3 weakly. Activation of these same G proteins by the ml MAChR was much less efficient, consistent with its known selectivity for pertussis toxin-insensitive G proteins ("Gp") that have not yet been isolated. The beta-AR and m2 MAChR were characteristically stimulated by reduction of disulfides. These results demonstrate the general utility of the baculovirus system for production of large quantities of native G protein-coupled receptors.

Adenylyl Cyclases↗
PubMed1990

A truncation mutation in the avian beta-adrenergic receptor causes agonist-induced internalization and GTP-sensitive agonist binding characteristic of mammalian receptors.

Recombinant turkey erythrocyte beta-adrenergic receptors expressed in murine L cells exhibited characteristic avian subtype selectivity for agonists and antagonists. In 10 of the 11 clones studied, no agonist-induced internalization of receptor was observed, although agonist-induced uncoupling of receptor and adenylyl cyclase occurred rapidly. GTP caused little or no decrease in affinity for beta-adrenergic agonists. Such behavior is commonly observed in avian erythrocytes. In contrast, one clone was susceptible to agonist-induced receptor internalization and down-regulation even though it exhibited characteristic avian beta-adrenergic ligand-binding properties. The affinity of this variant receptor for agonists was also notably reduced by GTP. Electrophoresis of affinity-labeled receptor from this clone indicated an apparent size of about 33 kDa, about 12 kDa less than that of the native or recombinant turkey beta-adrenergic receptor. Genomic DNA from this cell line that encodes the receptor was cloned and partially sequenced. The coding region of the original receptor cDNA was interrupted after codon 412 (out of 483) and was followed by 36 base pairs of novel sequence prior to the first in-frame stop codon. These results suggest that the lack of both hormone-induced internalization and GTP-sensitive, high affinity binding of agonists that is characteristic of the beta-adrenergic receptor in avian erythrocytes is due to intrinsic properties of the receptor. The restoration of these phenomena in a C-terminally truncated mutant receptor suggests the importance of the C-terminal domain in determining these processes.

Adenylyl Cyclases↗
PubMed1990

Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity.

Mastoparan (MP), a cationic, amphiphilic tetradecapeptide, stimulates guanine nucleotide exchange by GTP-binding regulatory proteins (G proteins) in a manner similar to that of G protein-coupled receptors. 1) MP stimulated exchange by isolated G protein alpha subunits and alpha beta gamma trimers. Relative stimulation was greater with alpha beta gamma trimers and beta gamma subunits could increase net MP-stimulated activity. 2) MP action was enhanced by reconstitution of trimeric G protein into phospholipid vesicles. Hill coefficients for activation were 2-4. The membrane-bound alpha-helical conformation of MP appeared to be the activating species. 3) MP blocked the ability of Go to increase the affinity of muscarinic receptors for agonist ligands, suggesting that MP and the receptor may compete for a common binding site on Go. 4) MP stimulated steady state GTPase activity at less than 1 microM Mg2+ and stimulated the dissociation of both GDP and guanosine 5'-O-(3-thiotriphosphate) at less than 1 nM Mg2+. Millimolar Mg2+ blocked the stimulatory effect of MP. Both high and low affinity Mg2+ binding sites are on the alpha subunit. 5) Increasing the amphiphilicity or hydrophobicity of MP enhanced its regulatory activity more than 2-fold and lowered the EC50 more than 10-fold. Several natural amphiphilic peptides also displayed modest stimulatory activity. 6) Benzalkonium chloride competitively antagonized the stimulation of Gi by MP but potently stimulated nucleotide exchange on Go. Because cationic, amphiphilic sequences on the cytoplasmic faces of receptors are required for G protein regulation, these findings suggest that nucleotide exchange on G proteins is regulated by the presentation of multiple cationic structures on the inner face of the plasma membrane.

Amines↗