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R Wallis

Publications and source records attributed to R Wallis.

At least 19 recordsLinked to original sources
PubMed2005

Impediments to policy implementation: the offer of free installation of central heating to an elderly community has limited uptake.

OBJECTIVES: Increasing the warmth of homes in the UK to combat excess winter mortality has become an important part of public policy. This study examined the success of a local initiative to provide free installation of central heating for a group of elderly people. STUDY DESIGN: Cohort study. METHODS: The uptake of the offer of central heating was monitored through its different stages. Where possible, comments were invited from the elderly tenants to whom the offer was made; a sub-sample was interviewed about their views. RESULTS: Of 1181 tenants approached, only around one-quarter finally received central heating within the year. Reasons for the low uptake included inaccuracies in estimating those eligible as well as tenants' worries over upheaval and mess, health concerns and fear of increased heating costs. CONCLUSION: Successful policy implementation requires not only command of appropriate financial resources and the backing of local 'implementers', but also the local resources with which the policy will engage, especially local knowledge, and the 'targets' of the policy initiative, in this case the elderly tenants.

Aged↗
PubMed2003

Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development.

OBJECTIVE: To attempt to determine the relative value of preclinical cardiac electrophysiology data (in vitro and in vivo) for predicting risk of torsade de pointes (TdP) in clinical use. METHODS: Published data on hERG (or I(Kr)) activity, cardiac action potential duration (at 90% repolarisation; APD(90)), and QT prolongation in dogs were compared against QT effects and reports of TdP in humans for 100 drugs. These data were set against the free plasma concentrations attained during clinical use (effective therapeutic plasma concentrations; ETPC(unbound)). The drugs were divided into five categories: (1) Class Ia and III antiarrhythmics; (2) Withdrawn from market due to TdP; (3) Measurable incidence/numerous reports of TdP in humans; (4) Isolated reports of TdP in humans; (5) No reports of TdP in humans. RESULTS: Data from hERG (or I(Kr)) assays in addition to ETPC(unbound) data were available for 52 drugs. For Category 1 drugs, data for hERG/I(Kr) IC(50), APD(90), QTc in animals and QTc in humans were generally close to or superimposed on the ETPC(unbound) values. This relationship was uncoupled in the other categories, with more complex relationships between the data. In Category 1 (except amiodarone), the ratios between hERG/I(Kr) IC(50) and ETPC(unbound) (max) ranged from 0.1- to 31-fold. Similar ranges were obtained for drugs in Category 2 (0.31- to 13-fold) and Category 3 (0.03- to 35-fold). A large spread was found for Category 4 drugs (0.13- to 35700-fold); this category embraced an assortment of mechanisms ranging from drugs which may well be affecting I(Kr) currents in clinical use (e.g. sparfloxacin) to others such as nifedipine (35700-fold) where channel block is not involved. Finally, for the majority of Category 5 drugs there was a >30-fold separation between hERG/I(Kr) activity and ETPC(unbound) values, with the notable exception of verapamil (1.7-fold), which is free from QT prolongation in man; this is probably explained by its multiple interactions with cardiac ion channels. CONCLUSIONS: The dataset confirms the widely-held belief that most drugs associated with TdP in humans are also associated with hERG K(+) channel block at concentrations close to or superimposed upon the free plasma concentrations found in clinical use. A 30-fold margin between C(max) and hERG IC(50) may suffice for drugs currently undergoing clinical evaluation, but for future drug discovery programmes, pharmaceutical companies should consider increasing this margin, particularly for drugs aimed at non-debilitating diseases. However, interactions with multiple cardiac ion channels can either mitigate or exacerbate the prolongation of APD and QT that would ensue from block of I(Kr) currents alone, and delay of repolarisation per se is not necessarily torsadogenic. Clearly, an integrated assessment of in vitro and in vivo data is required in order to predict the torsadogenic risk of a new candidate drug in humans.

Animals↗
PubMed2002

The effect of a fast bowling harness in cricket: an intervention study.

The influence of a bowling harness, as a training aid, was assessed as a means of modifying bowling technique. Thirty-three 13-year-old bowlers received a standardized 15 min of bowling coaching twice a week for 8 weeks. They were randomly assigned to one of two groups. The 13 participants in the intervention group used the bowling harness throughout the coaching, while also receiving verbal and visual feedback. The 20 participants in the non-harness group received the same visual and verbal feedback. Three-dimensional videography (200 Hz) of each player's bowling action enabled the calculation of transverse plane shoulder alignment counter-rotation, separation angle, lateral flexion and hyperextension of the trunk before and after the intervention. The restriction applied by the harness produced a significant reduction (P= 0.006) in separation angle and forced the bowler to adopt a position at back-foot impact that reduced the 'twist' in the spine. However, it had no effect on restricting other aspects of trunk movement during the critical phases of the bowling action. No significant long-term modifications to technique were found after the coaching intervention when players were assessed without the harness.

Adolescent↗
PubMed2002

The measurement of shoulder alignment in cricket fast bowling.

The aim of this study was to compare thoracic spine alignment with two- and three-dimensional calculations of shoulder alignment (defined as a line joining the acromion processes of the right and left scapula) when all measures were projected onto the transverse plane. A six-camera Vicon system was used to reconstruct three markers positioned on the plane of the thorax such that the orthogonal vector to the thoracic spine, projected onto the transverse plane, was used as a virtual shoulder alignment during cricket fast bowling. This same measurement system was used to calculate the three-dimensional line between the acromion processes projected onto the transverse plane. These acromion markers were also used to calculate the two-dimensional transverse plane alignment of the shoulders from images recorded by a video camera positioned above ball release. All cameras operated at 50 Hz. A significant association was recorded between thorax alignment and the three- (0.97) and two-dimensional (0.87) shoulder alignment estimations at back-foot impact. The strength of association remained at front-foot impact, when correlations of 0.89 (three-dimensional) and 0.84 (two-dimensional) were recorded. However, at ball release, non-significant associations of 0.58 (three-dimensional) and 0.41 (two-dimensional), representing shoulder alignment differences of approximately 10 degrees, were recorded. The 95% limits of agreement comparisons for shoulder alignment at back-foot impact, front-foot impact and ball release produced mean random errors for the two comparisons of 9.5 degrees, 11.7 degrees and 22.5 degrees respectively. Three- and two-dimensional transverse plane projections of shoulder alignment are reasonable estimates of thorax alignment at back-foot impact and front-foot impact but not at ball release.

Adolescent↗
PubMed2001

Stoichiometry of complexes between mannose-binding protein and its associated serine proteases. Defining functional units for complement activation.

Serum mannose-binding protein (MBP) initiates the lectin branch of the complement cascade by binding to sugars on the surfaces of microorganisms and activating two MBP-associated serine proteases (MASP-1 and MASP-2). Rat serum MBP consists of oligomers containing up to four copies of a subunit that is composed of three identical polypeptide chains. Biophysical analysis of intact and truncated MASPs indicates that each MASP is a homodimer that is stabilized through interactions involving an N-terminal CUB domain. The binding sites for MBP are formed from the three N-terminal MASP domains, in which two CUB modules interact with MBP. Each MASP dimer contains binding sites for two MBP subunits. Both sites must be occupied by subunits from a single MBP oligomer to form a stable complex. Thus, the smallest functional unit for complement activation consists of MBP dimers bound to MASP-1 or MASP-2 homodimers. Trimers and tetramers of MBP form complexes containing up to two MASPs. The results reveal how MASP-1 and MASP-2 can function independently to activate the complement cascade.

Carrier Proteins↗
PubMed2000

Interaction of mannose-binding protein with associated serine proteases: effects of naturally occurring mutations.

Mannose-binding protein (MBP; mannose-binding lectin) forms part of the innate immune system. By binding directly to carbohydrates on the surfaces of potential microbial pathogens, MBP and MBP-associated serine proteases (MASPs) can replace antibodies and complement components C1q, C1r, and C1s of the classical complement pathway. In order to investigate the mechanisms of MASP activation by MBP, the cDNAs of rat MASP-1 and -2 have been isolated, and portions encompassing the N-terminal CUB and epidermal growth factor-like domains have been expressed and purified. Biophysical characterization of the purified proteins indicates that each truncated MASP is a Ca(2+)-independent homodimer in solution, in which the interacting modules include the N-terminal two domains. Binding studies reveal that both MASPs associate independently with rat MBP in a Ca(2+)-dependent manner through interactions involving the N-terminal three domains. The biophysical properties of the truncated MASPs indicate that the interactions with MBP leading to complement activation differ significantly from those between components C1q, C1r, and C1s of the classical pathway. Analysis of MASP binding by rat MBP containing naturally occurring mutations equivalent to those associated with human immunodeficiency indicates that binding to both truncated MASP-1 and MASP-2 proteins is defective in such mutants.

Amino Acid Sequence↗
PubMed2000

Impaired secretion of rat mannose-binding protein resulting from mutations in the collagen-like domain.

Serum mannose-binding protein (MBP) or mannose-binding lectin initiates the lectin branch of the innate immune response by binding to the surface of potentially pathogenic microorganisms and initiating complement fixation through an N-terminal collagen-like domain. Mutations in this region of human MBP are associated with immunodeficiency resulting from a reduction in the ability of the mutant MBPs to fix complement as well as from reduced serum concentrations. Inefficient secretion of the mutant proteins, which is one possible cause of the reduced serum levels, has been investigated using a mammalian expression system in which each of the naturally occurring human mutations has been recreated in rat serum MBP. The mutations Gly25-->Asp and Gly28-->Glu disrupt the disulfide-bonding arrangement of the protein and cause at least a 5-fold increase in the half-time of secretion of MBP compared with wild-type rat serum MBP. A similar phenotype, including a 3-fold increase in the half-time of secretion, disruption of the disulfide bonding arrangement, and inefficient complement fixation, is observed when nearby glucosylgalactosyl hydroxylysine residues at positions 27 and 30 are replaced with arginine residues. The results suggest that defective secretion resulting from structural changes in the collagen-like domain is likely to be a contributory factor for MBP immunodeficiency.

Amino Acid Substitution↗
PubMed2000

Prospects for reducing and refining the use of dogs in the regulatory toxicity testing of pharmaceuticals.

A workshop was held to critically discuss the need for a nonrodent species and the role of the dog in regulatory toxicity testing of pharmaceuticals; to discuss opportunities to reduce and refine the use of dogs in preclinical toxicology; and to identify a number of specific recommendations which could be feasibly achieved to move the process forward. To facilitate a preliminary evaluation of the contribution of dog studies to the risk assessment process, anonymised, unpublished data were provided from fully evaluated, repeat-dose toxicity studies in the rat and dog. Results of the International Life Sciences Institute (ILSI) Human Toxicity Project were also presented and discussed. Analysis of the data demonstrated that the dog can provide additional toxicity information, which, in some cases, was shown to be predictive for humans. Discussions indicated that there is potential for achieving a reduction in dog use and several possible approaches were identified. To further the progress of this initiative, there is a need to collate the results of pharmacology, toxicology, and clinical studies to address some of the proposed approaches. One of the outcomes of the workshop will be the establishment of a steering group to co-ordinate data collation for further analysis.

Animal Testing Alternatives↗
PubMed2000

Post-anaesthetic shaking. A review of the literature.

This paper describes the phenomenon of post-anaesthetic shaking (PAS) as it is discussed in the literature. Literature was obtained via computerised searches of the Cochrane Library, Medline, and Cumulative Index of Nursing and Allied Health Literature (CINAHL) databases. This review considers PAS in adults, who have had either a general or regional anaesthetic. It focuses on the controversy regarding the cause of the condition, the role of anaesthetics in hypothermia, pharmacological interventions, and non-pharmacological interventions. The key conclusion to emerge is that nurses must take patients' shaking seriously and initiate treatment for it. If PAS is associated with hypothermia then the patient needs to be rewarmed, and if it is associated with pain, analgesia needs to be administered.

Humans↗
PubMed2000

Preventing post-anaesthetic shaking.

A number of post-operative patients experience uncontrollable shivering and shaking after regional or general anaesthesia (GA). As a recovery room nurse, I frequently observed this unpleasant phenomenon and wanted to find out more about it. The purpose of the research was twofold: to investigate the incidence of post-anaesthetic shaking in my own workplace and to develop a clinical tool for its treatment.

Anesthesia, Conduction↗
PubMed1999

Molecular defects in variant forms of mannose-binding protein associated with immunodeficiency.

Distinct molecular mechanisms underlying immunodeficiency caused by three different naturally occurring point mutations within the collagen-like domain of human mannose-binding protein (MBP; also known as mannose-binding lectin) have been revealed by introduction of analogous mutations into rat serum MBP. The change Arg23-->Cys results in a lower proportion of the large oligomers most efficient at activating the complement cascade. The presence of cysteine at position 23, which forms aberrant interchain disulfide bonds, causes disruption of the normal oligomeric state. The deficiency in MBPs containing Gly25-->Asp and Gly28-->Glu substitutions also results in part from reduced formation of higher oligomers. However, decreased ability to interact with downstream components of the complement cascade due to changes in both the N-terminal disulfide-bonding arrangement and the local structure of the collagenous domain make more important contributions to the loss of activity in these mutants.

Amino Acid Sequence↗
PubMed1999

Characterisation of the 5-HT receptor binding profile of eletriptan and kinetics of [3H]eletriptan binding at human 5-HT1B and 5-HT1D receptors.

The affinity of eletriptan ((R)-3-(1-methyl-2-pyrrolidinylmethyl)-5-[2-(phenylsulphonyl )ethyl]-1H-indole) for a range of 5-HT receptors was compared to values obtained for other 5-HT1B/1D receptor agonists known to be effective in the treatment of migraine. Eletriptan, like sumatriptan, zolmitriptan, naratriptan and rizatriptan had highest affinity for the human 5-HT1B, 5-HT1D and putative 5-ht1f receptor. Kinetic studies comparing the binding of [3H]eletriptan and [3H]sumatriptan to the human recombinant 5-HT1B and 5-HT1D receptors expressed in HeLa cells revealed that both radioligands bound with high specificity (>90%) and reached equilibrium within 10-15 min. However, [3H]eletriptan had over 6-fold higher affinity than [3H]sumatriptan at the 5-HT1D receptor (K(D)): 0.92 and 6.58 nM, respectively) and over 3-fold higher affinity than [3H]sumatriptan at the 5-HT1B receptor (K(D): 3.14 and 11.07 nM, respectively). Association and dissociation rates for both radioligands could only be accurately determined at the 5-HT1D receptor and then only at 4 degrees C. At this temperature, [3H]eletriptan had a significantly (P<0.05) faster association rate (K(on) 0.249 min(-1) nM(-1)) than [3H]sumatriptan (K(on) 0.024 min(-1) nM(-1)) and a significantly (P<0.05) slower off-rate (K(off) 0.027 min(-1) compared to 0.037 min(-1) for [3H]sumatriptan). These data indicate that eletriptan is a potent ligand at the human 5-HT1B, 5-HT1D, and 5-ht1f receptors and are consistent with its potent vasoconstrictor activity and use as a drug for the acute treatment of migraine headache.

Animals↗
PubMed1999

Characterisation of the contractile activity of eletriptan at the canine vascular 5-HT1B receptor.

The functional activity of eletriptan ((R)-3-(1-methyl-2-pyrrolidinylmethyl)-5-[2-(phenylsulphonyl )ethyl]- 1 H-indole) at the contractile serotonin (5-hydroxytryptamine; 5-HT) '1B-like' receptor in dog isolated saphenous vein and basilar artery was investigated. Eletriptan, like 5-HT and sumatriptan potently contracted saphenous vein (pEC50: 6.3, 6.9 and 6.1, respectively) and basilar artery (pEC50 7.2, 7.5 and 6.8, respectively). The maximum responses evoked by eletriptan was, unlike sumatriptan, significantly lower than that to 5-HT (intrinsic activity saphenous vein: eletriptan 0.57, 5-HT 1.0, sumatriptan 0.85; basilar artery: eletriptan 0.77, 5-HT 0.98, sumatriptan 0.89). Contractions evoked by eletriptan were antagonised by the 5-HT1B/1D receptor antagonist GR125743 (N-[4-methoxy-3-(4-methyl piperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide) with pA2 values of 9.1 in saphenous vein and 9.4 in basilar artery. Affinity estimates (pKA) for 5-HT and sumatriptan determined from receptor alkylation studies in saphenous vein were 6.6 and 6.3, respectively, compared to the apparent equilibrium dissociation constant (pKp) for eletriptan of 6.8. The rank order of relative intrinsic efficacies (epsilon) was 5-HT > sumatriptan > eletriptan. Thus, eletriptan required greater receptor occupancy (4.4-fold) to evoke an equivalent contraction to 5-HT and sumatriptan in dog isolated saphenous vein. These data demonstrate that eletriptan is a potent partial agonist at the canine vascular 5-HT1B receptor.

Alkylation↗
PubMed1999

Molecular determinants of oligomer formation and complement fixation in mannose-binding proteins.

Rat serum mannose-binding protein (MBP-A) functions as part of the innate immune system by targetting complement toward potentially pathogenic microorganisms. In order to examine the molecular basis for complement activation, rat MBP-A has been overproduced in Chinese hamster ovary cells. Recombinant protein is post-translationally modified in the same way as the native lectin. Hydrodynamic studies indicate that MBP-A consists predominantly of covalent oligomers containing one to four copies of a subunit that comprises a trimer of polypeptides. These oligomers are non-interconverting and do not assemble into higher order structures at concentrations in excess of those normally found in serum. Disulfide bonds formed between cysteine residues at the N-terminal end of the collagen-like domain link polypeptides to form covalent oligomers. Analysis of wild-type MBP-A and MBP-A containing the substitution Cys6 --> Ser suggests that polypeptides within each trimeric structural unit are mostly linked by disulfide bonds between cysteine residues at positions 13 and 18 arranged in an asymmetrical configuration. Disulfide bonds involving Cys6 connect polypeptides within separate trimers. Analysis of chimeras between MBP-A and rat liver MBP (MBP-C) indicates that residues within the N-terminal region of the collagenous domain and the cysteine-rich domain of MBP-A enable assembly of trimers into higher order oligomers. The activity of MBP-A in a hemolytic complement fixation assay using mannan-coated sheep erythrocytes was approximately 20-fold greater than the activity of MBP-C. Analysis of the MBP chimeras and isolated oligomers of MBP-A reveals that the larger oligomers are more efficient at complement activation. These data indicate that the overall complement fixing activity of MBP-A is a function of the individual molecular activities of oligomers and their relative abundance within the serum.

Amino Acid Sequence↗
PubMed1998

Enzymological characterization of the nuclease domain from the bacterial toxin colicin E9 from Escherichia coli.

The cytotoxicity of the bacterial toxin colicin E9 is due to a non-specific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin. CD spectroscopy shows the E9 DNase to be structured in solution, and analytical ultracentrifugation data indicate that the enzyme is a monomer. The nuclease activity of the E9 DNase was compared with the well-studied, non-specific DNase I by using a spectrophotometric assay with calf thymus DNA as the substrate. Both enzymes require divalent metal ions for activity but, unlike DNase I, the E9 DNase is not activated by Ca2+ ions. Somewhat surprisingly, the E9 DNase shows optimal activity and linear kinetics in the presence of transition metals such as Ni2+ and Co2+ but displays non-linear kinetics with metals such as Mg2+ and Ca2+. Conversely, Ni2+ and other transition metals showed poor activity in a plasmid-based nicking assay, yielding significant amounts of linearized plasmid, whereas Mg2+ was very active, with the main intermediate being open-circle DNA. The results suggest that, on entry into bacterial cells, the E9 DNase is likely to exhibit primarily Mg2+-dependent nicking activity against chromosomal DNA, although other metals could also be utilized to introduce both single- and double-strand cleavages.

Bacterial Proteins↗
PubMed1998

A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity.

We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity.

Amino Acid Sequence↗
PubMed1998

Specificity in protein-protein recognition: conserved Im9 residues are the major determinants of stability in the colicin E9 DNase-Im9 complex.

The endonuclease group of E colicins are a family of bacterial toxins whose cytotoxic activity in a producing host is inactivated by a specific immunity protein. The DNase of colicin E9 can be bound and inhibited by both cognate and noncognate immunity proteins, the dissociation constants for which span a range of 12-orders of magnitude. DNase binding specificity of the immunity proteins is governed primarily by helix II, the sequence of which is variable in this family of proteins. Heteronuclear NMR experiments have identified helix III along with helix II as the likely DNase binding site, although other regions of Im9 also showed perturbations on binding the E9 DNase. In the present work, we have used the NMR experiments as a guide for alanine scanning mutagenesis of Im9. Our data show that helices II and III of Im9 are indeed the DNase binding site and in addition quantitate the relative binding energy associated with each helix. We find that the conserved residues of helix III make the largest relative contribution toward E9 DNase binding. In conjunction with previous studies, the data suggest that specificity in the colicin-immunity system is governed by a dual recognition mechanism in which highly stabilizing interactions emanating from the conserved regions of an immunity protein act as the binding site anchor and these are modulated by interactions from neighboring, nonconserved amino acid residues. This modulation is likely to take the form of both favorable and unfavorable interactions, the balance of which define the specificity of the protein-protein interaction. The generality of such a dual recognition mechanism in other systems is also discussed.

Alanine↗