Restrict genetic susceptibility tests.
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Advisory Committees↗
Biomedical subjects
P Taylor
Publications and source records attributed to P Taylor.
Inhibition of glucose trimming with castanospermine reduces calnexin association and promotes proteasome degradation of the alpha-subunit of the nicotinic acetylcholine receptor.
To identify factors involved in the expression of ligand-gated ion channels, we expressed nicotinic acetylcholine receptors in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acetylcholine receptor traverse the membrane four times, contain at least one oligosaccharide, and are retained in the endoplasmic reticulum until completely assembled into the circular arrangement of subunits of delta-alpha-gamma-alpha-beta to enclose the ion channel. We previously demonstrated that calnexin is associated with unassembled subunits of the receptor, but appears to dissociate when subunits are assembled in various combinations. We used the glucosidase inhibitor castanospermine to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castanospermine treatment reduces the association of calnexin with the alpha-subunit of the receptor, and diminishes the intracellular accumulation of unassembled receptor subunit protein. However, treatment with castanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated alpha-subunits, and generally reverses the castanospermine induced loss of alpha-subunit protein. Co-transfection of cDNAs encoding the alpha- and delta-subunits, which leads to the expression of assembled alpha- and delta- subunits, also inhibits the loss of alpha-subunits expressed in the presence of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway.
Acetylcysteine↗
Public health. The missing link.
The government's strategy for public health may fail because of a lack of co-operation between organisations. Primary care, as presently constituted, has little energy to take on public health. The strategy places too much stress on the role of health authorities and too little on primary care and local communities. The predominance of the medical model in public health needs to be addressed.
Budgets↗
General practitioners' self assessment of knowledge. Testing can be valid only if questions are relevant to those tested.
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Education, Medical, Continuing↗
Distinctions in agonist and antagonist specificity conferred by anionic residues of the nicotinic acetylcholine receptor.
Two anionic residues in the nicotinic acetylcholine receptor, Asp-152 in the alpha-subunit and Asp-174 in the gamma-subunit or the corresponding Asp-180 in the delta-subunit, are presumed to reside near the two agonist binding sites at the alphagamma and alphadelta subunit interfaces of the receptor and have been implicated in electrostatic attraction of cationic ligands. Through site-directed mutagenesis and analysis of state changes in the receptor elicited by agonists, we have distinguished the roles of anionic residues in conferring ligand specificity and ligand-induced state changes. alphaAsp-152 affects agonist and antagonist affinity similarly, whereas gammaAsp-174 and deltaAsp-180 primarily affect agonist affinity. Combining charge neutralization on the alpha subunit with that on the gamma and delta subunits shows an additivity in free energy changes for carbamylcholine and d-tubocurarine, suggesting independent contributions of these residues to stabilizing the bound ligands. Since both aromatic and anionic residues stabilize cationic ligands, we substituted tyrosines (Y) for the aspartyl residues. While the substitution, alphaD152Y, reduced the affinities for agonists and antagonists, the gammaD174Y/deltaD180Y mutations reduced the affinity for agonist binding, but surprisingly enhanced the affinity for d-tubocurarine. To ascertain whether selective changes in agonist binding stem from the capacity of agonists to form the desensitized state of the receptor, carbamylcholine binding was measured in the presence of an allosteric inhibitor, proadifen. Mutant nAChRs carrying alphaD152Q or gammaD174N/deltaD180N show similar reductions in dissociation constants for the desensitized compared with activable receptor state and a similar proadifen concentration dependence. Hence, these mutations influence ligand recognition rather than the capacity of the receptor to desensitize. By contrast, the alphaD200Q mutation diminishes the ratio of dissociation constants for two states and requires higher proadifen concentrations to induce desensitization. Thus, the contributions of alphaAsp-152, gamma/deltaAsp-174/180, and alphaAsp-200 in stabilizing ligand binding can be distinguished by the interactions between agonists and allosteric inhibitors.
Amino Acid Sequence↗
Identification of pairwise interactions in the alpha-neurotoxin-nicotinic acetylcholine receptor complex through double mutant cycles.
alpha-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the alpha-neurotoxin from Naja mossambica mossambica (Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR alpha-subunit (Val188, Tyr190, Pro197, and Asp200) as contributing to binding. In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of alpha-neurotoxin from N. mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2-3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6-2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the alphadelta binding site with observed coupling energies of 1.5-1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two conserved cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are binding in close proximity to the alpha-subunit region between residues 188-200. The toxin residue Arg33 is closer to Val188, where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys27 is positioned closer to Tyr190, Pro197, and Asp200, where it is likely stabilized through electrostatic interaction with Asp200 and/or cation/pi interactions with Tyr190.
Amino Acid Sequence↗
Endocrine disruptors signal the need for receptor models and mechanisms to inform policy.
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Animals↗
An example of a protein ligand found by database mining: description of the docking method and its verification by a 2.3 A X-ray structure of a thrombin-ligand complex.
A computer program (SANDOCK) has been developed for the automated docking of small ligands to a target protein. It uses a guided matching algorithm to fit ligand atoms into the protein binding pocket. The protein is described by a modified Lee-Richard's dotted surface with each dot coded by chemical property and accessibility. Orientations of the ligand in the active site are generated such that a chemical and a shape complementary between the ligand and the active site cavity have to be fulfilled. The generated fits are evaluated with scoring functions which account for van der Waals, hydrophobic and hydrogen bonding interactions. This newly developed docking program can efficiently screen very large databases in a reasonable time and has been used to successfully identify novel ligands. The X-ray structure of a thrombin-ligand complex predicted by SANDOCK is described. The ligand binds to thrombin with a Kd of 65 microM and has an rmsd of 0.7 A for all ligand atoms from the predicted binding mode by SANDOCK.
Algorithms↗
The X-ray structure of a divergent cyclophilin from the nematode parasite Brugia malayi.
A structure of residues 1-177 of the cyclophilin domain of a large divergent cyclophilin from the filarial nematode parasite Brugia malayi has been crystallised and solved in two different crystal forms. The active site has a similar structure to that of human cyclophilin A. Two of the 13 residues important in forming the human cyclophilin A/cyclosporin A complex are altered in the B. malayi cyclophilin and explain the relatively poor inhibition of peptidyl prolyl isomerase activity by cyclosporin A.
Amino Acid Sequence↗
Corona stenting in a patient menorrhagia: use of a gonadotrophin releasing hormone agonist to facilitate anticoagulation.
We report a case with menorrhagia due to uterine fibroids who was admitted following an episode of unstable angina. Her coronary angiogram showed a tight proximal left anterior descending artery lesion for which stenting was deemed necessary. She was successfully anticoagulated in spite of her menorrhagia under cover of a gonadotrophin hormone releasing agonist; goserilin.
Angina, Unstable↗
An intensive weight loss programme in established type 2 diabetes and controls: effects on weight and atherosclerosis risk factors at 1 year.
The efficacy, safety, and effect on cardiovascular risk factors of two intensive weight loss programmes in overweight Type 2 diabetic subjects were studied. The patients were recruited from hospital diabetic clinics and control obese subjects from the community. Obese (BMI >30) patients with Type 2 diabetes mellitus and controls were offered intensive conventional diabetic advice or a very low calorie diet. Weekly 2 h sessions were conducted in two day-room areas of adjacent medical wards of Torbay Hospital. Non-diabetic and diabetic very low calorie diet groups reduced BMI by 6 and 5 kg m(-2), respectively, at 1 year. Waist-hip ratios (-0.06 and -0.05) were also reduced (p = 0.04 and p = 0.01), while HDL/total cholesterol ratios increased (+0.04 and +0.06, p = <0.01). Transient changes in blood pressure and antioxidant vitamin status occurred in the intensive conventional diet group. Fourteen of diabetic very low calorie diet subjects discontinued insulin and oral hypoglycaemic agents for the whole year, and psychological well-being transiently improved. Substantial weight loss and improvement in cardiovascular risk factors could be maintained for 1 year in Type 2 diabetic patients by the use of a very low calorie diet.
Adult↗
Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge.
It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.
Acetylcholinesterase↗
Biological insights from clinical trials with anti-TNF therapy.
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Antibodies, Monoclonal↗
Comparison of three monoclonal antibody pools for the detection of respiratory viral antigen in respiratory secretions.
The aim of this study was to compare the sensitivities of commercial monoclonal antibody pools to be used as an initial rapid screen for detection of viral antigens in respiratory secretions. The availability of commercial monoclonal antibodies has dramatically improved the detection of viruses by immunofluorescence techniques in exfoliated cells obtained from respiratory secretions. Several companies have recently introduced monoclonal antibody pools to detect the presence of respiratory viruses in a single preparation. Ninety-four stored slide preparations that had previously been examined by individual monoclonal antibodies were tested using three commercial monoclonal antibody pools produced by Sanofi (UK), Dako (UK), and Quadratech (UK). These monoclonal antibody pools had a sensitivity of 79.6%, 90.9%, and 100%, respectively, when compared with the original results. The overall intensity of immunofluorescence was also examined.
Antibodies, Monoclonal↗
Glycocalicin levels in the plasma of HIV+ patients: an indicator of platelet turnover.
Glycocalicin (GC) is the carbohydrate-rich portion of platelet membrane glycoprotein Ib(alpha) that can be cleaved from circulating platelets by proteases. The plasma GC level is an indicator of platelet turnover. Using an ELISA for GC, we assayed the plasma of 20 normal children (age 6 to 13 years), 50 HIV+ children (ages 4 to 18 years), 32 normal adults (ages 21 to 53 years), and 50 HIV+ adults (ages 24 to 66 years). The results were adjusted for individual platelet counts to give GC indexes (GCI). The normal children and the normal adults had significantly different GCI distributions (P = .002). In both normal and HIV+ individuals the GCI decreased with increasing platelet count (-.73 < r < -.34). Twenty-eight percent of the HIV+ children and 28% of the HIV+ adults had elevated GCI values. The majority of these elevated values occurred in patients with platelet counts >100,000/microL. Neither the GCI nor the platelet count was correlated with viral load. The platelet count, however, was weakly correlated with the CD4 count in both children (r = .31) and adults (r = .30) infected with HIV. Also, the CD4 count was weakly and inversely correlated with GCI in HIV+ adults (r = -.34) and in children (r = -.24). We conclude that increased GCI and, by implication, increased platelet turnover is a relatively common feature of advanced HIV disease. Furthermore, GCI may be elevated in HIV+ patients even with a platelet count >100,000/microL, suggesting increased platelet turnover before thrombocytopenia develops.
Adolescent↗
Nociceptive and antinociceptive responses to intrathecally administered nicotinic agonists.
Activation of spinal nicotinic receptors evokes a prominent algogenic response. Recently, epibatidine, a potent nicotinic agonist, was found to display an antinociceptive response after systemic administration. To examine the spinal component of this action, effects of three nicotinic agonists epibatidine, cytisine and nicotine--were given intrathecally (IT) and their antinociceptive activity was subsequently assessed. All agents elicited dose-dependent algogenic activity, characterized at lower doses by touch-evoked hyperactivity and at higher doses by intermittent vocalization and marked behavioral activity, with the rank order of potency being epibatidine > cytisine > nicotine. In addition, intrathecal epibatidine elicited a short-lasting, dose-dependent thermal antinociception. In contrast, the other nicotinic agonists at the highest usable dose failed to produce a significant antinociception. Mecamylamine, a nicotinic channel blocker, completely abolished the antinociceptive and algogenic responses of epibatidine. The competitive antagonist, alpha-lobeline, blocked both the analgesic and algogenic responses, but methyllycaconitine inhibited only the algogenic effects of epibatidine. Dihydro-beta-erythroidine, also a competitive antagonist, had no effect on the initial intense algogenic responses. The analgesic response to epibatidine was neither inhibited by naloxone nor by atropine. 2-Amino-5-phosphopentanoic acid, a competitive N-methyl-D-aspartate receptor antagonist, did not affect the analgesic response to intrathecal epibatidine or the intense initial algogenic response. Upon repeated administration (30-min interval), epibatidine (1 microg, IT) exhibited marked and rapid desensitization to both the analgesic and algogenic responses which recovered within 8 h. Pretreatment with two consecutive doses of cytisine (5 microg, IT, 30-min apart) inhibited the agitation and analgesic actions of intrathecal epibatidine. Thus, we contend that in addition to the typical nociceptive response elicited by spinal nicotinic agonists, intrathecal epibatidine also exhibits a pronounced but short-lasting antinociception. The analgesic and algogenic responses to intrathecal epibatidine may be mediated by distinct subtypes of spinal nicotinic receptors as suggested by the antagonist studies.
Alkaloids↗
Inhibition of mouse acetylcholinesterase by fasciculin: crystal structure of the complex and mutagenesis of fasciculin.
Fasciculins are members of the superfamily of three-fingered peptidic toxins from Elapidae venoms. They selectively inhibit mammalian and electric fish acetylcholinesterases (AChE) with Ki values in the pico- to nanomolar range. Kinetic studies performed in solution indicate that fasciculin does not totally occlude ligand access to the active site of AChE, but rather binds to a peripheral site of the enzyme to inhibit catalysis, perhaps allosterically. The crystal structure of the Fas2-mouse AChE complex delineated a large contact area consistent with the low dissociation constant of the complex; the Fas2 and AChE residues participating in the binding interface were unambiguously established, and major hydrophobic interactions were identified. The structure however suggests that fasciculin totally occludes substrate entry into the catalytic site of AChE, and does not reveal to what extent each contact between Fas2 and AChE contributes to the overall binding energy. New probes, designed to delineate the individual contributions of the fasciculin residues to the complex formation and conformation, were generated by site-directed mutagenesis of a synthetic Fas2 gene. A fully processed recombinant fasciculin, rFas2, that is undistinguishable from the natural, venom-derived Fas2, was expressed in a mammalian system; fourteen mutants, encompassing 16 amino acid residues distributed among the three loops (fingers) of Fas2, were developed from both the kinetic and structural data and analyzed for inhibition of mouse AChE. The determinants identified by the structural and the functional approaches do coincide. However, only a few of the many residues which make up the overall interactive site of the Fas2 molecule provide the strong interactions required for high affinity binding and enzyme inhibition. Potential drug design from the fasciculin molecule is discussed.
Acetylcholine↗