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A Wise

Publications and source records attributed to A Wise.

At least 37 records · Page 2Linked to original sources
PubMed1999

Appropriate uses for spreadsheets, databases and statistical software for the analysis of dietary data.

The article presents a new approach to analysis of dietary data, emphasizing the use of database systems, but including spreadsheets and statistical programs in the process. Spreadsheets are useful in dietary research for calculations required prior to entry of the data into a dietary analysis program, for investigation of the possible effects of changing dietary components and for graphical representation of data. A dietary program can provide output of data for a large number of subjects' diets into a database. Databases should be increasingly used in data analysis due to their powerful capacity to provide immediate answers to complex research questions. The database can be used to provide output directly in a form that can be pasted into a statistical program. Some knowledge of structured query language is required to construct appropriate queries of the database; an introduction to this language with some examples is given in this article.

Databases, Factual↗
PubMed1999

A new strategy for controlling the level of activation in artificially stimulated muscle.

Distributed stimulation of slow skeletal muscle has previously been used to produce smooth tetanic contractions at low stimulus rates. This involved distributed or interleaved stimulation of portions of the muscle with near equal tension contributions. Extending this to fast and mixed muscle encounters difficulties in getting and maintaining equal twitch responses for the portions. This need has now been circumvented by using distributed stimulation with unequal interpulse intervals. Described here is a microprocessor-based eight channel distributed muscle stimulator that can adjust stimulation timing to produce an optimally smooth tension over a range of stimulus rates even when the portions are unequal. This design is based on modeling results. Distributed stimulation experiments performed on skeletal muscle show that this method can be used to achieve smooth tension at physiological stimulus rates, which should reduce fatigue. This has important implications in functional neuromuscular stimulation (FNS) as well as in enabling experiments to be conducted to characterize the biomechanical behavior of partially activated fast and mixed muscle.

Animals↗
PubMed1999

The amino terminus of receptor activity modifying proteins is a critical determinant of glycosylation state and ligand binding of calcitonin receptor-like receptor.

The calcitonin receptor-like receptor (CRLR) can function as either a receptor for calcitonin gene-related peptide (CGRP) or for adrenomedullin (ADM), depending upon the coexpression of a novel family of single transmembrane proteins, which we have called receptor activity modifying proteins or RAMPs. RAMPs 1, 2, and 3 transport CRLR to the plasma membrane with similar efficiencies, however RAMP1 presents CRLR as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMPs 2 and 3 present CRLR as an immature, core glycosylated ADM receptor. Characterization of the RAMP2/CRLR and RAMP3/CRLR receptors in HEK293T cells by radioligand binding (125I-ADM as radioligand), functional assay (cAMP measurement), or biochemical analysis (SDS-polyacrylamide gel electrophoresis) revealed them to be indistinguishable, even though RAMPs 2 and 3 share only 30% identity. Chimeric proteins were created with the transmembrane and cytosolic portions of RAMP1 associated with the amino terminus of RAMP2 (RAMP2/1) and vice versa (RAMP1/2). Coexpression of RAMP2/1 with CRLR formed a core glycosylated ADM receptor, whereas the RAMP1/2 chimera generated both core glycosylated and mature forms of CRLR and enabled both ADM and CGRP receptor binding. Hence, the glycosylation state of CRLR appears to correlate with its pharmacology.

Adrenomedullin↗
PubMed1998

Heterodimerization is required for the formation of a functional GABA(B) receptor.

GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABA(A/C)) receptors to produce fast synaptic inhibition and metabotropic (GABA(B)) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABA(B) receptor (GABA(B)R1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABA(B)R2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABA(B)R2-receptor subtype forms heterodimers with GABA(B)R1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABA(B)R2 in HEK293T cells, GABA(B)R1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABA(B) receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABA(B) receptors may be heterodimers composed of GABA(B)R1 and GABA(B)R2.

Amino Acid Sequence↗
PubMed1998

Hydrophobicity of residue351 of the G protein Gi1 alpha determines the extent of activation by the alpha 2A-adrenoceptor.

Cysteine351 is the site for pertussis toxin-catalyzed ADP-ribosylation in the G protein Gi1 alpha. Alteration of this residue, or the equivalent cysteine in other Gi-family G proteins, has been used to examine specific interactions between receptors and these G proteins. However, no systematic analysis has been performed to determine the quantitative effect of such alterations. To address this we mutated cysteine351 of Gi1 alpha to all other possible amino acids. Each of the G protein mutants was transiently coexpressed along with the porcine alpha 2A-adrenoceptor in HEK 293/T cells. Following pertussis toxin treatment of the cells, membranes were prepared and the capacity of the agonist UK14304 to stimulate the binding of [35S]GTP gamma S to the modified G proteins was measured. A spectrum of function was observed. The presence of either a charged amino acid or a proline at this position essentially attenuated agonist regulation. The wild-type G protein did not result in maximal stimulation by agonist. The presence of certain branched chain aliphatic amino acids or bulky aromatic R groups at amino acid351 resulted in substantially greater maximal stimulation by the alpha 2A-adrenoceptor than that achieved with the wild-type sequence. The degree of activation of the forms of Gi1 alpha correlated strongly with the octanol/water partition coefficient of the amino acid at residue351. Variation in EC50 values for agonist-induced stimulation of binding of [35S]GTP gamma S to the mutant G proteins also correlated with the octanol/water partition coefficient. These results define a central role for hydrophobicity of this residue in defining productive receptor-G protein interactions.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1998

RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor.

Calcitonin-gene-related peptide (CGRP) and adrenomedullin are related peptides with distinct pharmacological profiles. Here we show that a receptor with seven transmembrane domains, the calcitonin-receptor-like receptor (CRLR), can function as either a CGRP receptor or an adrenomedullin receptor, depending on which members of a new family of single-transmembrane-domain proteins, which we have called receptor-activity-modifying proteins or RAMPs, are expressed. RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents the receptor at the cell surface as a mature glycoprotein and a CGRP receptor. RAMP2-transported receptors are core-glycosylated and are adrenomedullin receptors.

3T3 Cells↗
PubMed1998

Quantitative analysis of a cysteine351glycine mutation in the G protein Gi1alpha: effect on alpha2A-adrenoceptor-Gi1alpha fusion protein activation.

Fusion proteins were constructed between the porcine alpha2A-adrenoceptor and either wild-type (Cys351) or a pertussis toxin-resistant (Gly351) form of the G protein Gi1alpha. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration-dependent stimulation of their high affinity GTPase activity. The alpha2A-adrenoceptor-wild type Gi1alpha fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly351 Gi1alpha. Treatment of the fusion proteins as agonist-regulated enzymes allowed measurement of Vmax and turnover number for adrenaline-stimulation of the GTPase activity of each fusion construct. The turnover number of the alpha2A-adrenoceptor-Cys351 Gly Gi1alpha fusion protein was only 44'S, of that for the alpha2A-adrenoceptor-wild type Gi1alpha fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist-occupied receptor to activate the mutant.

Animals↗
PubMed1998

Agonist occupation of an alpha2A-adrenoreceptor-Gi1alpha fusion protein results in activation of both receptor-linked and endogenous Gi proteins. Comparisons of their contributions to GTPase activity and signal transduction and analysis of receptor-G protein activation stoichiometry.

A fusion protein between a pertussis toxin-resistant (C351G) mutant of the alpha subunit of the G protein Gi1 and the porcine alpha2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts. Agonists caused stimulation of high affinity GTPase activity, which was partially prevented by pertussis toxin treatment, demonstrating that the toxin-resistant component of the GTPase activity was derived from the receptor-fused G protein and the remainder from endogenous Gialpha. Half-maximal stimulation of the GTPase activity of endogenous Gi was achieved with lower concentrations of agonist. Although the Km for GTP of the fusion protein-linked Gi was lower than for the endogenous G protein, Vmax measurements demonstrated that adrenaline activated some 5 mol of endogenous Gi/mol of fusion protein-linked Gi. The isolated alpha2A-adrenoreceptor could activate Gs; however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous Gialpha was greater than for the fusion protein-constrained C351G Gi1alpha. alpha2A-Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated alpha2A-adrenoreceptor-C351G Gi1alpha fusion protein-expressing cells, but these signals were abolished following pertussis toxin treatment. These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1998

Alpha-noradrenergic receptor modulation of the phencyclidine- and delta9-tetrahydrocannabinol-induced increases in dopamine utilization in rat prefrontal cortex.

The noncompetitive NMDA receptor antagonist phencyclidine (PCP) and the neuronal cannabinoid receptor agonist delta9-tetrahydrocannabinol (THC) are two agents shown to have psychotomimetic properties in humans. Both drugs increase dopamine release and utilization in the prefrontal cortex, a brain region thought to be dysfunctional in schizophrenia. In the present series of studies, the effects of drugs acting at alpha-noradrenergic receptors on PCP- and THC-induced increases in prefrontal cortical and nucleus accumbens dopamine utilization in the rat were examined. Clonidine, an alpha2 noradrenergic receptor agonist, completely blocked the activation of mesoprefrontal dopamine system by THC or PCP. In addition, the alpha1 noradrenergic receptor antagonist prazosin blocked the PCP-induced increase in prefrontal cortical dopamine utilization. These data may provide new insights concerning pharmacological therapies for acute drug-induced psychoses and behavioral abnormalities in human PCP and THC abusers.

Adrenergic alpha-Agonists↗
PubMed1998

Child and adolescent schizophrenia.

Until only very recently, the occurrence of schizophrenia in childhood and early adolescence had been largely neglected. Improved diagnostic formulations have resulted in clarification of the boundaries between childhood schizophrenia, other psychotic disorders, and pervasive developmental disorder. The study of schizophrenia in childhood or adolescence provides a unique opportunity to examine illness characteristics in the absence of the confounds of substance abuse illness, chronicity, and medication effects. Additionally, current etiopathologic models of schizophrenia can be tested in this patient subgroup.

Adolescent↗
PubMed1997

Measurement of agonist efficacy using an alpha2A-adrenoceptor-Gi1alpha fusion protein.

A fusion protein was constructed between the porcine alpha2A-adrenoceptor and a pertussis toxin-insensitive (Cys351Gly) form of the alpha subunit of the G protein Gi1. Addition of agonist ligands to membranes of COS-7 cells transiently transfected to express this construct, and treated with pertussis toxin prior to cell harvest, resulted in stimulation of both high affinity GTPase activity and enhanced binding of [35S]GTPgammaS. By considering the fusion protein as an agonist-activated enzyme and measuring Vmax of GTP hydrolysis a range of agonist ligands displayed varying efficacy in their capacity to activate the receptor-associated G protein with adrenaline = noradrenaline = alpha-methylnoradrenaline > UK14304 > BHT933 > or = xylazine = clonidine. A similar rank order was observed following independent co-expression of the alpha2A-adrenoceptor and Cys351Gly-Gi1alpha. These data demonstrate the utility and applicability of using a receptor-G protein fusion protein approach, in which the stoichiometry of receptor and G protein is fixed at 1:1, to measure and further understand the nature of agonist efficacy.

Adrenergic alpha-Agonists↗
PubMed1997

Rescue of functional interactions between the alpha2A-adrenoreceptor and acylation-resistant forms of Gi1alpha by expressing the proteins from chimeric open reading frames.

Co-expression of the alpha2A-adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the alpha subunit of Gi1 resulted in agonist-induced, pertussis toxin-independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G Gi1alpha generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G Gi1alpha rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) alpha2A-adrenoreceptor and either C351G or C3S/C351G Gi1alpha also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of Gi1alpha are cytosolic proteins. Expression of these as chimeric alpha2A-adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission.

Acylation↗
PubMed1997

A cysteine-3 to serine mutation of the G-protein Gi1 alpha abrogates functional activation by the alpha 2A-adrenoceptor but not interactions with the beta gamma complex.

Pertussis toxin-resistant (C351G) and also palmitoylation-negative (C3S/C351G), myristoylation-negative (G2A/C351G) and combined acylation-negative (G2A/C3S/C351G) forms of the G-protein Gi1 alpha were expressed in COS-7 cells along with the porcine alpha 2A-adrenoceptor. G2A/C3S/C351G Gi1 alpha and G2A/C351G Gi1 alpha were largely cytosolic and failed to interact with the agonist-occupied alpha 2A-adrenoceptor in membrane preparations. In contrast, C351G Gi1 alpha was almost entirely particulate and the alpha 2-adrenoceptor agonist UK14304 caused a marked stimulation of its GTPase activity and binding of [35S]GTP gamma S which was not prevented by pertussis toxin treatment of the cells. C3S/C351G Gi1 alpha was present in both the particulate and cytosolic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the alpha 2A-adrenoceptor. Coexpression of C3S/C351G Gi1 alpha and the alpha 2A-adrenoceptor along with beta 1 and gamma 2 subunits increased the P2 membrane complement of the alpha subunit and increased substantially the ratio of membrane bound to cytosolic protein. However, this also failed to allow marked stimulation of high-affinity GTPase activity by the alpha 2A-adrenoceptor despite the increased proportion of G-protein in the P2 membrane fraction. Despite the low fractional activation of C3S/C351G Gi1 alpha by the alpha 2A-adrenoceptor compared to C351G Gi1 alpha, the palmitoylation-resistant G-protein caused a marked reduction in pertussis toxin-resistant, agonist (UK14304)-mediated stimulation of adenylyl cyclase activity. UK14304 caused the same degree of effect on adenylyl cyclase activity in pertussis toxin-treated cells following transfection of the same amounts of C351G Gi1 alpha and C3S/C351G Gi1 alpha, as both appear to act to sequester beta gamma subunits. By contrast, neither G2A/C351G Gi1 alpha nor G2A/C3S/C351G Gi1 alpha resulted in effective regulation of adenylyl cyclase activity.

Adenylate Cyclase Toxin↗
PubMed1997

Measurement of agonist-induced guanine nucleotide turnover by the G-protein Gi1alpha when constrained within an alpha2A-adrenoceptor-Gi1alpha fusion protein.

A fusion protein was generated between the porcine alpha2A-adrenoceptor and a pertussis-toxin-insensitive (Cys351-->Gly) variant of the alpha subunit of Gi1alpha by direct in-frame fusion of the N-terminus of the G-protein to the C-terminus of the receptor. The fusion protein could be transiently expressed to high levels in COS-7 cells. Addition of the alpha2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro- 1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) to membranes of pertussis-toxin-treated transfected cells resulted in a concentration-dependent stimulation of high-affinity GTPase activity. Vmax estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0+/-0.2 min-1 for Gi1alpha when the alpha2A-adrenoceptor was maximally stimulated by UK14304 with a Km for GTP of 0.37+/-0.07 microM. Co-expression of excess beta1gamma2 along with the alpha2A-adrenoceptor-Gi1alpha fusion protein resulted in greater maximal UK14304-induced stimulation of high-affinity GTPase activity (2.1+/-0.2-fold) without alteration in agonist EC50. These studies demonstrate the functionality of the fusion construct, its capacity to interact with betagamma complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.

Adrenergic alpha-Agonists↗
PubMed1997

Interaction of the G-protein G11alpha with receptors and phosphoinositidase C: the contribution of G-protein palmitoylation and membrane association.

Wild-type and palmitoylation defective mutants of the murine G protein G11alpha were transfected into HEK293 cells. Wild-type G11alpha was membrane associated, Cys9Ser Cys10Ser G11alpha was present in the soluble fraction whilst both Cys9Ser G11alpha and Cys10Ser G11alpha were distributed between the fractions. Expression of the rat TRH receptor resulted in agonist stimulation of inositol phosphate accumulation. The degree of stimulation produced by TRH following co-transfection of the palmitoylation-resistant forms of G11alpha compared to the wild-type protein correlated with the amount of membrane-associated G protein.

Animals↗
PubMed1997

Interactions of the alpha2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants.

The alpha2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'Gi-like' pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein alpha-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1alpha, Gi2alpha and Gi3alpha resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys --> Gly mutations of Gi1alpha, Gi2alpha and Gi3alpha were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys --> Gly mutants of Gi1alpha, Gi2alpha and Gi3alpha required 10-15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the alpha2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.

Adenylate Cyclase Toxin↗
PubMed1997

Expression of the Arabidopsis G-protein GP alpha1: purification and characterisation of the recombinant protein.

The Arabidopsis G alpha subunit, GP alpha1, was expressed within Escherichia coli by co-transformation with the expression vector and the dnaY gene which encodes tRNA(Arg)(AGA/AGG) Isolation of the recombinant GP alpha1 in a highly pure form could be achieved by a combination of anion exchange and dye affinity chromatography or by a single step affinity procedure via chromatography on 4-amino-anilido-GTP agarose. The recombinant protein yielded by both procedures was highly active and bound GTPgammaS with an apparent Kd in the nM range. GTPgammaS binding was stimulated two-fold in the presence of Zn2+ compared with that in the presence of Mg2+, Mn2+ or Ca2+.

Arabidopsis↗