The case against a national screening programme for aortic aneurysms.
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Aortic Aneurysm, Abdominal↗
Biomedical subjects
R J Williams
Publications and source records attributed to R J Williams.
Hypolipidaemic properties of a potent and bioavailable alkylsulphinyl-diphenylimidazole ACAT inhibitor (RP 73163) in animals fed diets low in cholesterol.
RP 73163 ((S)-2-[5-(3,5-dimethyl-l-pyrazolyl)pent-l-yl)-sulphinyl]-5, 6-diphenylimidazole) has been shown to be a potent and specific inhibitor of acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26; ACAT) in vitro using the tissues of experimental animals as sources of the enzyme. The concentrations of RP 73163 required to produce 50% inhibition of ACAT activity (IC50 values) in microsomal preparations ranged from 86 nM for rat liver to 370 nM for rabbit intestine. In whole cell assays using human hepatic (HepG2), intestinal (Caco2), and monocytic (THP-1) cell lines, RP 73163 inhibited ACAT activity with IC50 values of 266, 158, and 314 nM, respectively. The addition of RP 73163 (0.03-1.0 microM) to the medium of cultured HepG2 cells produced a concentration-dependent decrease in apolipoprotein B (apoB) secretion. The compound has high systemic bioavailability. Using a bioassay, a concentration of active inhibitor equivalent to 29 microM of parent compound was present in plasma 1 hr after oral administration of RP 73163 (50 mg.kg-1). In rats that had been fed a basal diet ad libitum or starved for 18 hr prior to blood sampling, the administration of RP 73163 (50 mg.kg-1 b.i.d. for 7 days) reduced plasma triglyceride levels by 50% without affecting the concentration of cholesterol. This hypotriglyceridaemic effect was associated with reductions in plasma very-low-density-lipoprotein (VLDL) and low-density-lipoprotein (LDL) levels. RP 73163 decreased the rate of VLDL secretion by 24% in Triton WR-1339-treated rats that had been fasted overnight but did not affect the secretion rate in animals fed ad libitum, indicating that ACAT was only important in regulating VLDL secretion under certain nutritional conditions. RP 73163 reduced the accumulation of intraperitoneally administered [3H]leucine into the plasma VLDL-apoB pool in both fed and fasted states. The results suggest that, in fed animals at least, an increase in the clearance of VLDL from the bloodstream may contribute to the hypolipidaemic activity of the compound. In rabbits with casein-induced endogenous hypercholesterolaemia, RP 73163 specifically reduced the levels of cholesterol carried by LDL. In conclusion, the hypolipidaemic actions of RP 73163, a potent and systemically bioavailable ACAT inhibitor, are consistent with a reduction in the secretion of apoB containing lipoproteins by hepatic tissue and possibly with an increase in the clearance of these particles.
Animals↗
Acyl-CoA:Cholesterol O-acyltransferase (ACAT) inhibitors. 2. 2-(1,3-Dioxan-2-yl)-4,5-diphenyl-1H-imidazoles as potent inhibitors of ACAT.
The second in this series of papers concerns our further investigations into the search for a potent bioavailable acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor suitable for the treatment of atherosclerosis. The design, synthesis, and structure-activity relationship for a series of ACAT inhibitors based on the 2-(1,3-dioxan-2-yl)-4,5-diphenyl-1H-imidazole pharmacophore are described. Compounds such as 13a bearing simple alkyl or hydroxymethyl substituents at the 5-position of the 1,3-dioxane ring are potent bioavailable inhibitors of the rat hepatic microsomal enzyme in vitro (IC50 < 100 nM) but are only weak inhibitors of the human hepatic enzyme. We have found however that 1,3-dioxanes substituted at the 5-cis position with pyrazolylalkyl or aminoalkyl groups are potent inhibitors in vitro of human macrophage ACAT, the potency depending on the nature of the terminal heterocycle and the length of the alkyl chain. An ex vivo bioassay herein demonstrates that potent inhibitors such as 13t (IC50 = 10 nM) which contain lipophilic terminal heterocycles do not appear to be systematically available. Less potent but more water soluble compounds such as 13h (IC50 = 60 nM) and 13n (IC50 = 70 nM) are absorbed following oral dosing and achieve plasma levels significantly in excess of their IC50 for ACAT inhibition. These compounds are therefore possible candidates for further investigation as oral antiatherosclerotic agents.
Animals↗
RP 64477: a potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase with low systemic bioavailability.
RP 64477 (N-butyl-3-(p-decyloxybenzamido)-4-(methylthio)benzamide) has been shown to be a potent inhibitor of the cholesterol esterifying enzyme Acyl-coenzyme A:cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) in intestinal, hepatic, adrenal, and arterial tissue preparations obtained from a range of animal species. Drug concentrations producing 50% inhibition of enzyme activity (IC50 values) ranged from 14-283 nM. Inhibition by RP 64477 in a rabbit intestinal enzyme preparation was shown to be non-competitive with respect to the substrate oleoyl-CoA. In whole cell assays using human intestinal (CaCo-2), hepatic HepG2) and monocytic (THP-1) cell lines, RP 64477 inhibited ACAT activity with IC50s of 113, 503, and 180 nM, respectively. RP 64477 (0.03% w/w by diet) reduced significantly cholesterol absorption in cholesterol/cholic acid-fed rats from 94+/- 8% to 65 +/- 4%. In cholesterol-fed rabbits, cholesterol absorption was reduced from 72 +/- 5% to 50 +/-5% and 44 +/- 5% at dose levels of 10 and 30 mg kg-1 b.i.d., respectively. Plasma cholesterol levels were reduced dose-dependently in both cholesterol/cholic-acid-fed rats and cholesterol-fed rabbits. Neither cholesterol absorption nor plasma cholesterol levels were reduced significantly in animals maintained on standard laboratory diets. Pharmacokinetic studies indicated that RP 64477 were very poorly absorbed following oral administration to rats. Plasma levels of drug were < 2 ng mL-1 following a dose of 2000 mg kg-1 p.o.. When radiolabelled RP 64477 was administered orally, limited absorption was indicated by the overwhelming elimination of radioactivity in the faces (96.4% of administered material) coupled with low renal clearance (0.6% of dose) and biliary excretion (0.05% of dose). In conclusion, this work shows that RP 64477 is a potent inhibitor of ACAT obtained from a range of animal species and man. Inhibition of cholesterol absorption and hypocholesterolaemic activity has been demonstrated in rats and rabbits maintained on diets supplemented with cholesterol. Pharmacokinetic studies indicate low systemic exposure to RP 64477 as a result of limited absorption of this drug.
Acyl Coenzyme A↗
Retinal artery embolization: a rare presentation of calcific aortic stenosis.
Retinal artery embolization is an unusual but serious complication of calcific aortic stenosis. However, it is rare for retinal embolization to be the presenting feature of aortic stenosis. This report describes a young patient who presented with an acute retinal artery occlusion secondary to calcific aortic valve disease, and discusses the rationale for early surgical intervention.
Acute Disease↗
Rat to human extrapolation of HCFC-123 kinetics deduced from halothane kinetics: a corollary approach to physiologically based pharmacokinetic modeling.
The goal of this study was to develop a human physiologically based pharmacokinetic (PBPK) model for the chemical HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane) and its major metabolite, trifluoroacetic acid (TFA). No human kinetic data for HCFC-123 are available, thus a corollary approach was developed. HCFC-123 is a structural analog of the common anesthetic agent halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) and follows a common pathway of oxidative biotransformation, resulting in the formation of the same metabolite, TFA. In this study, halothane models for rats and humans were developed and validated. Then the corollary approach was used to develop a human HCFC-123 model from a rat HCFC-123 model. This strategy was implemented by using a previously validated PBPK model for HCFC-123/TFA in the Fisher 344 rat as a template model for halothane in rats. Model predictions were then compared to, and were in good agreement with, measured values for the concentration of halothane in rat blood and fat tissue. A human PBPK model for halothane was developed. The identical mode structure (with the exception of the description for the fat compartment) that was used to describe halothane and TFA in the rat was used for describing halothane and TFA in the human. Human physiological parameters for tissue volumes and flows were taken from the literature, and human tissue partition coefficients for halothane were measured in the laboratory. Based on reported similarity in metabolism of halothane by humans and rats, metabolic constants for halothane in the rat were used in the human model, and specific parameters describing the kinetics of TFA were estimated by optimization. The model was validated against human exposure data for halothane from six published studies (expired breath concentrations of halothane and serum/urine data for TFA). A similar approach was then used to derive a human HCFC-123 model for humans from the HCFC-123 rat model. The corollary approach described here illustrates the innovative use of template model structures to aid in the development and validation of models for structural analogs with similar metabolism and activity in biologic systems. Furthermore, given that the PBPK model adequately describes the kinetics of halothane in rats and humans and of HCFC-123 in rats, use of the human PBPK model is proposed for deriving dose-response estimates of human health risks in the absence of human kinetic data.
Animals↗
Reduced glucose metabolism enhances the glutamate-evoked release of arachidonic acid from striatal neurons.
Glucose deprivation potentiates the glutamate receptor-evoked release of arachidonic acid from cultured mouse striatal neurons. In this study we investigated whether this potentiation would be modified by the end-products of glycolysis. These enhanced responses were completely reversed by the addition of increasing concentrations of either lactate or pyruvate. This reversal was not due to increased osmolarity as substituting sucrose for lactate or pyruvate did not mimic their effects. In contrast, in the presence of glucose, neither lactate nor pyruvate was effective. Furthermore, these monocarboxylic acids rescued neuronal respiration in the absence of glucose. Inhibiting glycolysis with iodoacetate in the presence of glucose reproduced the potentiated glutamate-evoked release of arachidonic acid observed following glucose deprivation and reduced neuronal respiration to the same extent as that observed in the absence of glucose. All of these effects were overcome by the addition of either lactate or pyruvate. The reversal of the potentiated glutamate-evoked release of arachidonic acid by lactate or pyruvate was inhibited by a specific inhibitor of monocarboxylic acid transport, alpha-cyano-4-hydroxycinnamic acid, suggesting that lactate and pyruvate act intracellularly. Therefore, we propose that the enhanced release of arachidonic acid evoked by glutamate during glucose deprivation results from reduced glycolysis and hence from a depletion of lactate or pyruvate.
Animals↗
Calcium binding proteins in normal and transformed cells, Perugia, May 1996.
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Calcium-Binding Proteins↗
Cyclothiazide unmasks an AMPA-evoked release of arachidonic acid from cultured striatal neurones.
The joint, but not independent, activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and metabotropic glutamate receptors induces liberation of arachidonic acid from cultured mouse striatal neurones. We examined whether blocking AMPA receptor desensitisation with cyclothiazide would modify this response. Cyclothiazide strongly potentiated the combined AMPA/(1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-evoked release of arachidonic acid (EC50 of approximately 7 microM) but did not modulate the basal, ACPD, or NMDA response. The enhanced liberation of arachidonic acid, observed in the presence of cyclothiazide, was due to the appearance of a genuine AMPA response that was independent of an associative activation of metabotropic receptors. The potentiated and nonpotentiated AMPA responses were inhibited by both competitive [2,3-di-hydroxy-6-nitro-7-sulphamoylbenzo (f) quinoxaline] and 2,3-benzodiazepine noncompetitive (GYKI 53655 and GYKI 52466) receptor antagonists. Cyclothiazide was equally effective at potentiating the AMPA response in either the presence or absence of glucose, suggesting that the increased glutamate-evoked arachidonic acid release observed in these cells under conditions of glucose deprivation is not due to reduced AMPA receptor desensitisation. The enhanced liberation of arachidonic acid measured in the presence of cyclothiazide appeared to result from a large (fourfold) elevation of the AMPA-induced increase in intracellular calcium level. Therefore, an AMPA-evoked mobilisation of arachidonic acid could potentially contribute to non-NMDA receptor-mediated neurotoxicity, which has been observed in neuronal cells in the presence of cyclothiazide.
Animals↗
Arthroscopic release of the posterior cruciate ligament for stiff total knee arthroplasty.
Ten posterior cruciate ligament sparing total knee arthroplasties were studied in 9 patients who underwent arthroscopic posterior cruciate ligament release to improve intractable postoperative knee stiffness and pain. Mean patient age was 64 years. Before posterior cruciate ligament release, the mean knee extension was 4 degrees (0 degrees - 10 degrees); mean knee flexion was 73.9 degrees (40 degrees - 110 degrees). The mean interval to posterior cruciate ligament release after primary total knee arthroplasty was 29 months. After release, mean extension was 1.3 degrees (0 degrees - 10 degrees) and mean flexion was 112 degrees (range, 85 degrees - 135 degrees). The average increase in flexion observed in the immediate postoperative period was 40.1 degrees (25 degrees - 60 degrees). At a mean followup of 20 months, mean extension was 1.5 degrees (0 degrees - 10 degrees), and mean flexion was 104.5 degrees (70 degrees - 125 degrees). At followup, the average increase in knee flexion was 30.5 degrees (10 degrees - 50 degrees). Before arthroscopic posterior cruciate ligament release, the average Knee Society knee and function scores were 70.9 and 71, respectively. At followup, the average Knee Society knee and function scores were 86.4 and 88, respectively. The Knee Society pain score improved from 33.5 preoperatively to 42 postoperatively. All patients reported subjective improvements in pain and stiffness. Eight patients (88%) reported satisfaction with the procedure. Two patients (22%) eventually required revision total knee arthroplasty.
Adult↗
Leiomyoma of the hand in a child who has the human immunodeficiency virus.
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Child↗
Expectancies relate to symptoms of alcohol dependence in young adults.
A significant relationship between the Alcohol Dependence Scale (ADS, Skinner & Allen, 1982) and positive expectancies for alcohol use (AEQ, Brown et al., 1987) is reported that has implications for educational programmes designed to promote sensible drinking. Expectancies predicted current drinking and dependence scores among a sample of young adults that contained 14.6% of subjects scoring above the cut-off point on the ADS. AEQ scores were found to predict ADS scores over and above current drinking and the AEQ subscales successfully differentiated between heavy drinking and dependent drinking. Canonical correlations revealed that for males higher scores on Increased social assertiveness, Sexual enhancement and Arousal and power were related to higher symptoms of loss of control over drinking. The picture was more complex for females in that three canonical variates were found. First, personal expectancies elevated across all the subscales of the AEQ were related to Loss of control. Secondly, females with high scores on Global positive changes and Sexual enhancement showed more symptoms of obsessive drinking and finally, females with higher Arousal and power expectancies but lower Sexual enhancement also had higher scores on the Perceptual withdrawal subscale from the ADS.
Adult↗
Energised (entatic) states of groups and of secondary structures in proteins and metalloproteins.
In this review I have examined the functional value of selected states of isolated groups in proteins energised away from their expected ground states whether they are observed with or without energy perturbation of larger parts of a protein structure. These energisations, found in the absence of substrates, are called 'entatic states of groups' [Vallee, B. L. & Williams, R. J. P. (1968) Proc. Natl Acad. Sci. USA 59, 498-505]. A group can be part of an amino acid or any bound metal ion or cofactor. In some particular cases the apoprotein, where the energised metal ion or cofactor has been removed, or the protein in which the energised amino acid has been replaced, has the same back-bone structure as the holoprotein and even side-chains are only slightly adjusted. This case is quite different from a condition of a group simultaneously energised with a protein fold, due to their combination, and which therefore involves conformational change in the protein and the group and which may adjust the group while the protein tightens. The final condition is again stable but removal of the group now must result in a reversed protein conformation change. This simultaneous energisation of both the adjustable protein and the group can be local, as in an induced fit or more extensive when it can be likened in some cases to a stretching by rack action, or may involve a change from an almost random to a structured protein when the group is energised in a very limited way. The various energisations must not be confused since they differ functionally. The first can give rise to optimal heightened catalytic (or other functional) potential of the local group but cannot be connected either to excitation of other parts of a protein as in induced fitting, or to a relay of energy (larger conformational change) in the protein. Clearly it restricts the rate of exchange of a group. Induced fit can also give rise to group activation, though to a somewhat reduced degree, while increasing exchange rate. A device such as a rack may rather give rise to a mechanical activity (message transmission), which is relayed a large distance into the protein, and can only give considerably lower activation of individual groups but exchange may now be fast. The final case involves very modest energisation of the group with gross rearrangement and energisation of the protein and may be associated with storage or carrier functions. The groups upon which I concentrate are metal ions since detailed electronic and structural knowledge of their ground states are well known, allowing energised states to be easily detected, but the ideas apply equally to organic side-chains of proteins as will be shown. A further energisation can arise from the addition of a substrate to each kind of protein. In fact all the ideas of energisation applicable to groups having cyclic activity in permanent features of protein structure are equally well applied to substrate binding or conversion of substrates through excited states to products.
Apoproteins↗
Solution characterisation by NMR spectroscopy of two horseradish peroxidase isoenzyme C mutants with alanine replacing either Phe142 or Phe143.
Site-directed mutagenesis of the horseradish peroxidase isoenzyme C (HRP C) gene has been undertaken in order to provide two recombinant enzymes where alanine replaces either Phe142 or Phe143 ([F142A]HRP C and [F143A]HRP C, respectively). These heme enzymes have been characterised in solution using proton NMR spectroscopy for both the high-spin resting and low-spin cyanide-ligated states. Comparison of their NMR spectra with those recorded for wild-type plant HRP C indicates that both the protein fold and the structure of the heme pocket are maintained. The structural integrity of the aromatic donor molecule binding site is altered as a result of the substitution of Phe142 by Ala, but not by the corresponding substitution at Phe143. This is evident from analysis of perturbations to the chemical shift and linewidth parameters of the proton resonances of two Phe side chains, Phe A and Phe B, that participate in this site. The resting and cyanide-ligated states of [F142A]HRP C bind the aromatic donor molecule, benzhydroxamic acid, three to four times more weakly than the analogous states of wild-type plant HRP C. A titration of cyanide-ligated [F142A]HRP C with benzhydroxamic acid, monitored by NMR spectroscopy, further reveals that the dynamics of complex formation are considerably altered, in that only one of the two possible benzhydroxamic acid binding modes established for the cyanide-ligated wild-type enzyme is significantly populated. Although the assignment of Phe A and Phe B cannot be made to either Phe142 or Phe143, the results confirm that Phe142 is an important, although indirect, determinant of aromatic donor molecular binding and dynamics. The role of phenylalanine side chains in the binding of aromatic donor molecules by heme peroxidases is discussed in the light of these observations and a recent structural model for HRP C.
Alanine↗
Conformational studies of osteocalcin in solution.
1H-NMR and circular dichroism studies have been carried out on osteocalcin, a 49-residue, calcium-binding protein, the sequence of which contains a disulphide bridge, a proline-rich segment and three gamma-carboxyglutamic acid (Gla) residues. These latter residues have been proposed to lie on one face of an alpha helix and interact with the mineral phase, leading to incorporation of the protein into the bone matrix. Circular dichroism shows an increase in the alpha-helical structure on Ca2+ binding to bovine osteocalcin. This induced structure is lost on heating the protein, giving a spectrum close to that of the Ca(2+)-free protein. 1H-NMR studies of rabbit osteocalcin gave a set of resonance assignments and NOEs which could be interpreted in terms of distance constraints. These did not allow a single conformation to be defined for the protein in solution but reflect rather a flexible structure which may be essential for the function of the protein. The calculated structures contain a hydrophobic core (comprising Leu2, Leu32, Val36 and Tyr42, seen to be slowly flipping in the Ca(2+)-bound form) and have the gamma-carboxyglutamic acid side chains exposed on one face of the molecule.
Amino Acid Sequence↗
Bioenergetics. Purpose of proton pathways.
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Electron Transport↗
Identification of two new genes in the Pseudomonas aeruginosa amidase operon, encoding an ATPase (AmiB) and a putative integral membrane protein (AmiS).
The nucleotide sequence of the amidase operon of Pseudomonas aeruginosa has been completed and two new genes identified amiB and amiS. The complete gene order for the operon is thus amiEBCRS. The amiB gene encodes a 42-kDa protein containing an ATP binding motif that shares extensive homology with the Clp family of proteins and also to an open reading frame adjacent to the amidase gene from Rhodococcus erythropolis. Deletion of the amiB gene has no apparent effect on inducible amidase expression and it is thus unlikely to encode a regulatory protein. A maltose-binding protein-AmiB fusion has been purified and shown to have an intrinsic ATPase activity (Km = 174 +/- 15 mM; Vmax = 2.4 +/- 0.1 mM/min/mg), which is effectively inhibited by ammonium vanadate and ADP. The amiS gene encodes an 18-kDa protein with a high content of hydrophobic residues. Hydropathy analysis suggests the presence of six transmembrane helices in this protein. The AmiS sequences is homologous to an open reading frame identified adjacent to the amidase gene from Mycobacterium smegmatis and to the ureI gene from the urease operon of Helicobacter pylori. AmiS and its homologs appear to be a novel family of integral membrane proteins. Together AmiB and AmiS resemble two components of an ABC transporter system.
Adenosine Triphosphatases↗
The use of methyl-substituted benzhydroxamic acids as structural probes of peroxidase substrate binding.
The interactions between cyanide-ligated horseradish peroxidase (isoenzyme C) and a series of monosubstituted methylbenzhydroxamic acids have been studied using proton NMR techniques, and compared with analogous data for benzhydroxamic acid itself. Use of the methyl-substituted compounds in two-dimensional NOE experiments provides additional data on the structure of the aromatic donor molecule binding site through identification of new NOE connectivities with amino acid side-chains in the heme binding pocket. In this context the methyl group of the substituted benzhydroxamic acids acts as an effective probe of their local environment in the aromatic donor molecule binding site. While all the benzhydroxamic acids interact strongly with the heme edge at heme methyl C18H3, the methyl groups of 3-methylbenzhydroxamic and 4-methylbenzhydroxamic acids principally interact with the distal His42 C epsilon 1H and Phe A side-chains respectively. This confirms that the binding site is situated towards the distal side of the heme plane and that the Phe A side-chain known to participate in aromatic donor binding is located towards the entrance of the site. Dynamic properties of these peroxidase complexes are addressed in terms of ligand exchange phenomena and a comparison made with data available for benzhydroxamic acid. The existence of alternative binding modes in the case of the methylbenzhydroxamic acids is also discussed.
Horseradish Peroxidase↗