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Biomedical subjects

J Hirst

Publications and source records attributed to J Hirst.

At least 19 recordsLinked to original sources
PubMed2005

Energy transduction by respiratory complex I--an evaluation of current knowledge.

Complex I (NADH:ubiquinone oxidoreductase) is a membrane-bound, multisubunit enzyme. At present, there is no high-resolution structural model available for complex I and its mechanism of energy transduction is unknown. However, the subunit compositions of complex I from both eukaryotic and prokaryotic organisms have been determined, the sequences of their subunits are known and a coherent picture of the redox cofactors present in complex I has been developed. Here, I aim to describe and examine data relating to the mechanism of complex I and to build a framework to facilitate the discussion of possible conclusions and mechanistic models.

Animals↗
PubMed2005

Effects of intraperitoneal injection of Rofecoxib in a mouse model of ALS.

There is increasing evidence that inflammatory mechanisms are involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Inhibition of a key mediator of inflammation, cyclooxygenase 2 (COX-2), represents a promising therapeutic approach in ALS. Here we tested the in vivo effects of a specific COX-2 inhibitor, Rofecoxib, administered by intraperitoneal injection, in the SOD1(G93A G1H) mouse model of the familial form of ALS (fALS). Rofecoxib administration commenced at postnatal day 60 (P60), since the hallmarks of inflammation in the spinal cord were found to occur beyond this time-point in this mouse model of fALS. We found a significant but small delay in the onset of locomotor impairment in mice treated with Rofecoxib at the dose of 10 mg/kg of weight. However, survival was not effected by treatment. As prostaglandin E2 levels in spinal cord or in plasma were not reduced by Rofecoxib treatment, these results may suggest lack of sufficient bioavailability as the reason for the modest clinical changes observed.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Myometrial activation and preterm labour: evidence supporting a role for the prostaglandin F receptor--a review.

An increase in the myometrial expression of the prostaglandin (PG) receptors, and especially the PGF(2alpha) receptor (FP), may be an important component of the process initiating preterm labour. In this review of the literature and presentation of new possibilities, evidence will be discussed that demonstrates an increase in mouse uterine FP mRNA occurs at preterm birth whereas uterine PGF(2alpha) concentrations do not increase, suggesting elevated uterine receptor expression and sensitivity is a mechanism for preterm labour initiation. The first examination of the complete human myometrial FP promoter will be described and evidence presented that demonstrates the pro-inflammatory cytokine, interleukin-1beta, stimulates FP mRNA expression. Finally new data showing that administration of a specific FP antagonist delays preterm birth in sheep will be presented.

Animals↗
PubMed2001

GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).

The sequences of 42 subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria have been described previously. Seven are encoded by mitochondrial DNA, whereas the remaining 35 are nuclear gene products imported into the organelle from the cytoplasm. An additional protein, which does not correspond to any previously known subunit of the complex I assembly, has now been detected. Denaturing gels of subcomplex Ilambda, the hydrophilic arm of complex I, clearly show a hitherto unidentified band, which was digested with trypsin and subjected to mass-spectrometric analysis to provide several peptide sequences, used in cDNA cloning and sequencing. Measurement of the intact protein mass indicated that the N terminus is acetylated. The new complex I subunit (B16.6) is the bovine homolog of GRIM-19, the product of a cell death regulatory gene induced by interferon-beta and retinoic acid, thus providing a new link between the mitochondrion and its electron-transport chain and apoptotic cell death.

Amino Acid Sequence↗
PubMed2001

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 1. Effects on structure.

Replacement of the axial histidine ligand with exogenous imidazole has been accomplished in a number of heme protein mutants, where it often serves to complement the functional properties of the protein. In this paper, we describe the effects of pH and buffer ion on the crystal structure of the H175G mutant of cytochrome c peroxidase, in which the histidine tether between the heme and the protein backbone is replaced by bound imidazole. The structures show that imidazole can occupy the proximal H175G cavity under a number of experimental conditions, but that the details of the interaction with the protein and the coordination to the heme are markedly dependent on conditions. Replacement of the tethered histidine ligand with imidazole permits the heme to shift slightly in its pocket, allowing it to adopt either a planar or distally domed conformation. H175G crystallized from both high phosphate and imidazole concentrations exists as a novel, 5-coordinate phosphate bound state, in which the proximal imidazole is dissociated and the distal phosphate is coordinated to the iron. To accommodate this bound phosphate, the side chains of His-52 and Asn-82 alter their positions and a significant conformational change in the surrounding protein backbone occurs. In the absence of phosphate, imidazole binds to the proximal H175G cavity in a pH-dependent fashion. At pH 7, imidazole is directly coordinated to the heme (d(Fe--Im) = 2.0 A) with a nearby distal water (d(Fe--HOH) = 2.4 A). This is similar to the structure of WT CCP except that the iron lies closer in the heme plane, and the hydrogen bond between imidazole and Asp-235 (d(Im--Asp) = 3.1 A) is longer than for WT CCP (d(His--Asp) = 2.9 A). As the pH is dropped to 5, imidazole dissociates from the heme (d(Fe--Im) = 2.9 A), but remains in the proximal cavity where it is strongly hydrogen bonded to Asp-235 (d(Im--Asp) = 2.8 A). In addition, the heme is significantly domed toward the distal pocket where it may coordinate a water molecule. Finally, the structure of H175G/Im, pH 6, at low temperature (100 K) is very similar to that at room temperature, except that the water above the distal heme face is not present. This study concludes that steric restrictions imposed by the covalently tethered histidine restrain the heme and its ligand coordination from distortions that would arise in the absence of the restricted tether. Coupled with the functional and spectroscopic properties described in the following paper in this issue, these structures help to illustrate how the delicate and critical interactions between protein, ligand, and metal modulate the function of heme enzymes.

Amino Acid Substitution↗
PubMed2001

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function.

The inability of imidazole to complement function in the axial histidine deletion mutant, H175G, of yeast cytochrome c peroxidase has been an intriguing but unresolved issue that impacts our understanding of the role of axial ligands in heme catalysis. Here we report the functional and spectroscopic properties of H175G and of its complexes with imidazole. Combined with the crystal structures for these complexes, the data provide a detailed and consistent account of the modes of Im binding in the H175G cavity and their dependence on buffer and pH. UV--vis, EPR, and resonance Raman spectra reveal multiple coordination states for H175G/Im which can be correlated with the crystal structures to assign the following heme environments: H175G/H(2)O/H(2)O, H175G/Im(d)/phosphate(c), H175G/Im(d)/H(2)O(c), H175G/Im(c)/H(2)O(d), and H175G/Im(c)/OH(-)(c), where H175G/X/Y defines the proximal species as X and the distal species as Y and c and d subscripts refer, where known, to the coordinated and dissociated states, respectively. Resonance Raman data for reduced H175G/Im show two substates for heme-coordinated Im differing in the strength of their hydrogen bond to Asp-235, in a fashion similar to WT CCP. NO binding to ferrous H175G/Im results in dissociation of Im from the heme but not from the cavity, while no dissociation is observed for WT CCP, indicating that steric tethering may, in part, control NO-induced dissociation of trans ligands. H175G/Im forms an oxidized compound I state with two distinct radical species, each with a dramatically different anisotropy and spin relaxation from that of the Trp-191 radical of WT CCP. It is suggested that these signals arise from alternate conformations of Trp191 having different degrees of exchange coupling to the ferryl heme, possibly mediated by the conformational heterogeneity of Im within the H175G cavity. The kinetics of the reaction of H175G/Im with H(2)O(2) are multiphasic, also reflecting the multiple coordination states of Im. The rate of the fastest phase is essentially identical to that of WT CCP, indicating that the H175G/Im(c)/H(2)O(d) state is fully reactive with peroxide. However, the overall rate of enzyme turnover using cytochrome c as a substrate is <5% of WT and is unaffected by Im coordination. In summary, Im coordination to H175G results in a number of conformers, one of which is structurally and spectroscopically very similar to WT CCP. However, while this form is fully reactive with peroxide, the reaction with cytochrome c remains inefficient, perhaps implicating the altered Trp-191 radical species.

Amino Acid Substitution↗
PubMed2001

GGAs: roles of the different domains and comparison with AP-1 and clathrin.

We have previously identified a novel family of proteins called the GGAs (Golgi-localized, gamma-ear-containing, ADP-ribosylation factor-binding proteins). These proteins consist of an NH(2)-terminal VHS domain, followed by a GAT domain, a variable domain, and a gamma-adaptin ear homology domain. Studies from our own laboratory and others, making use of both yeast and mammals cells, indicate that the GGAs facilitate trafficking from the trans-Golgi network to endosomes. Here we have further investigated the function of the GGAs. We find that GGA-deficient yeast are not only defective in vacuolar protein sorting but they are also impaired in their ability to process alpha-factor. Using deletion mutants and chimeras, we show that the VHS domain is required for GGA function and that the VHS domain from Vps27p will not substitute for the GGA VHS domain. In contrast, the gamma-adaptin ear homology domain contributes to GGA function but is not absolutely required, and full function can be restored by replacing the GGA ear domain with the gamma-adaptin ear domain. Deleting the gamma-adaptin gene together with the two GGA genes exacerbates the phenotype in yeast, suggesting that they function on parallel pathways. In mammalian cells, the association of GGAs with the membrane is extremely unstable, which may account for their absence from purified clathrin-coated vesicles. Double- and triple-labeling immunofluorescence experiments indicate that the GGAs and AP-1 are associated with distinct populations of clathrin-coated vesicles budding from the trans-Golgi network. Together with results from other studies, our findings suggest that the GGAs act as monomeric adaptors, with the four domains involved in cargo selection, membrane localization, clathrin binding, and accessory protein recruitment.

ADP-Ribosylation Factors↗
PubMed2001

Pakistani and indigenous "white" women's views and the Donabedian-Maxwell grid: a consumer-focused template for assessing the quality of maternity care.

A criticism of using women's views as an assessment of the maternity services in the UK is that the views of some have been under-represented and inappropriately researched, in particular those from minority ethnic groups. In addition, there is criticism that "what" has been assessed within the maternity services, and other health-care arenas, has been driven by the perspective of those who provide the service rather than those who use it. The purpose here is to explore integrating Pakistani and indigenous "white" women's views with the Donabedian-Maxwell grid, as a means of offering a consumer-focused template for underpinning quality assessment and quality assurance strategies for the maternity services in the UK.

Arabs↗
PubMed2000

Atomically defined mechanism for proton transfer to a buried redox centre in a protein.

The basis of the chemiosmotic theory is that energy from light or respiration is used to generate a trans-membrane proton gradient. This is largely achieved by membrane-spanning enzymes known as 'proton pumps. There is intense interest in experiments which reveal, at the molecular level, how protons are drawn through proteins. Here we report the mechanism, at atomic resolution, for a single long-range electron-coupled proton transfer. In Azotobacter vinelandii ferredoxin I, reduction of a buried iron-sulphur cluster draws in a solvent proton, whereas re-oxidation is 'gated' by proton release to the solvent. Studies of this 'proton-transferring module' by fast-scan protein film voltammetry, high-resolution crystallography, site-directed mutagenesis and molecular dynamics, reveal that proton transfer is exquisitely sensitive to the position and pK of a single amino acid. The proton is delivered through the protein matrix by rapid penetrative excursions of the side-chain carboxylate of a surface residue (Asp 15), whose pK shifts in response to the electrostatic charge on the iron-sulphur cluster. Our analysis defines the structural, dynamic and energetic requirements for proton courier groups in redox-driven proton-pumping enzymes.

Aspartic Acid↗
PubMed2000

A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome.

We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH(2)-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal "ear" domain of gamma-adaptin. However, unlike gamma-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the gamma-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.

ADP-Ribosylation Factors↗
PubMed2000

Unusual oxidative chemistry of N(omega)-hydroxyarginine and N-hydroxyguanidine catalyzed at an engineered cavity in a heme peroxidase.

Heme enzymes are capable of catalyzing a range of oxidative chemistry with high specificity, depending on the surrounding protein environment. We describe here a reaction catalyzed by a mutant of cytochrome c peroxidase, which is similar but distinct from those catalyzed by nitric-oxide synthase. In the R48A mutant, an expanded water-filled cavity was created above the distal heme face. N-hydroxyguanidine (NHG) but not guanidine was shown to bind in the cavity with K(d) = 8.5 mM, and coordinate to the heme to give a low spin state. Reaction of R48A with peroxide produced a Fe(IV)=O/Trp(.+) center capable of oxidizing either NHG or N(omega)-hydroxyarginine (NHA), but not arginine or guanidine, by a multi-turnover catalytic process. Oxidation of either NHG or NHA by R48A did not result in the accumulation of NO, NO(2)(-), NO(3)(-), urea, or citrulline, but instead afforded a yellow product with absorption maxima of 257 and 400 nm. Mass spectrometry of the derivatized NHA products identified the yellow species as N-nitrosoarginine. We suggest that a nitrosylating agent, possibly derived from HNO, is produced by the oxidation of one molecule of substrate. This then reacts with a second substrate molecule to form the observed N-nitroso products. This complex chemistry illustrates how the active sites of enzymes such as nitric-oxide synthase may serve to prevent alternative reactions from occurring, in addition to enabling those desired.

Arginine↗
PubMed2000

Directional coronary atherectomy in intermediate sized vessels: final results of the intermediate vessel atherectomy trial (IVAT).

Revascularization options for intermediate sized vessels (2.5-2.9 mm) have historically been limited. IVAT is a pilot study to assess the efficacy and safety of debulking intermediate sized vessels using directional coronary atherectomy (DCA). Between March 1996 and June 1997, 50 patients were enrolled at seven hospitals in the United States. Of those patients, 70% presented with unstable angina and 52% had single vessel disease. Of the lesions treated, 96% were de novo. Adjunctive PTCA after DCA was performed in 90% of cases at the discretion of the investigator to maximize luminal diameter. The GTO DCA device was used in 90% of cases. Procedural success (residual stenosis <50% without major complications) was 94%. Stents were placed in 12% of patients. The only complications were three non-Q wave MIs. Mean reference vessel diameter increased from 2.49 mm pre-procedure to 2.57 mm after DCA and 2.61 post-procedure; mean MLD increased from 0.76 mm to 2.03 mm to 2.31 mm; and mean stenosis decreased from 70% to 21% post DCA and to 11% post procedure. At six months follow-up, 18.0% of target lesions required revascularization. Total revascularization, including non-target vessels, was 32%. These results suggest that DCA has a high procedural success rate and a low target lesion revascularization rate in intermediate sized vessels.

Adult↗
PubMed2000

Analysis of single-nucleotide polymorphisms in the interleukin-4 receptor gene for association with inflammatory bowel disease.

Genetic linkage analysis in families with multiple cases of inflammatory bowel disease (IBD) has mapped a gene which confers susceptibility to IBD to the pericentromeric region of chromosome 16 (IBD1). The linked region includes the interleukin(IL)-4 receptor gene (IL4R). Since IL-4 regulation and expression are abnormal in IBD, the IL4R gene is thus both a positional and functional candidate for IBD1. We screened the gene for single-nucleotide polymorphisms (SNPs) by fluorescent chemical cleavage analysis, and tested a subset of known and novel SNPs for allelic association with IBD in 355 families, which included 435 cases of Crohn's disease and 329 cases of ulcerative colitis. No association was observed between a haplotype of four SNPs (val50ile, gln576arg, A3044G, G3289A) and either the Crohn's disease or ulcerative colitis phenotypes using the transmission disequilibrium test. There was also no evidence for association when the four markers were analyzed individually. The results indicate that these variants are not significant genetic determinants of IBD, and that the IL4R gene is unlikely to be IBD1. Linkage disequilibrium analyses showed that the val50ile and gln576arg variants are in complete equilibrium with each other, although they are separated by only about 21 kilobases of genomic DNA. This suggests that a very dense SNP map may be required to exclude or detect disease associations with some candidate genes.

Alleles↗
PubMed2000

Fast voltammetric studies of the kinetics and energetics of coupled electron-transfer reactions in proteins.

A wealth of information on the reactions of redox-active sites in proteins can be obtained by voltammetric studies in which the protein sample is arranged as a layer on an electrode surface. By carrying out cyclic voltammetry over a wide range of scan rates and exploiting the ability to poise or pulse the electrode potential between cycles, data are obtained that are conveniently (albeit simplistically) analysed in terms of plots of peak potentials against scan rate. A simple reversible electron-transfer process gives rise to a 'trumpet'-shaped plot because the oxidation and reduction peaks separate increasingly at high scan rate; the electrochemical kinetics are then determined by fitting to Butler-Volmer or Marcus models. Much more interesting though are the ways in which this 'trumpet plot' is altered, often dramatically, when electron transfer is coupled to biologically important processes such as proton transfer, ligand exchange, or a change in conformation. It is then possible to derive particularly detailed information on the kinetics, energetics and mechanism of reactions that may not revealed clearly or even at all by other methods. In order to interpret the voltammetry of coupled systems, it is important to be able to define 'ideal behaviour' for systems that are expected to show simple and uncoupled electron transfer. Accordingly, this paper describes results we have obtained for several proteins that are expected to show such behaviour, and compares these results with theoretical predictions.

Electrochemistry↗
PubMed2000

Antenatal home blood pressure monitoring: a pilot randomised controlled trial.

OBJECTIVE: To measure recruitment to, compliance with, and the acceptability of a trial designed to test whether a reduced schedule of antenatal visits combined with training in self-measurement of blood pressure at home may improve hypertension screening and save money. To test the specific hypothesis that even after taking into account extra unscheduled visits, the reduced schedule with ambulatory monitoring reduces total visits. DESIGN: A pilot randomised controlled trial. SETTING: Four urban and four rural general practices in Yorkshire and Lancashire. POPULATION: One hundred and five low risk women in the third trimester of pregnancy. Eighty women participated. INTERVENTION: Women were invited to participate at 24-28 weeks. Those who accepted were allocated either to a standard nine subsequent visit schedule (30, 32, 34, 36, 37, 38, 39, 40, 41 weeks) or to a reduced schedule (34, 38, 41 weeks). Those in the latter group measured their blood pressure weekly using a portable sphygmomanometer at home. MAIN OUTCOME MEASURES: Recruitment, total number of clinic visits, frequency of blood pressure measurement, schedule preference, and anxiety. RESULTS: Although there were more unscheduled visits in the home monitoring group, this did not outweigh the reduction in scheduled visits, (total visits reduced from 7 x 4 to 4 x 5, P < 0 x 001), and blood pressure was measured during more weeks (9 vs 7 weeks, P < 0 x 001) in the experimental group. Most women expressed a preference for the reduced schedule both when the idea was first suggested, and after they had experienced it, and there were no significant differences in anxiety. CONCLUSION: Replacement of antenatal screening visits with home blood pressure monitoring is acceptable to women. The reduction in clinic visits is not compensated by an increase in visits for other reasons and overall blood pressure measurement is omitted less often. Whether it reduces adverse outcomes or has any rare side effects will require a larger trial, but this pilot study indicates that it is likely to be safe, and that such a large trial would be feasible.

Adult↗