Search PubMed⌕ Search

Biomedical subjects

A Reilly

Publications and source records attributed to A Reilly.

At least 37 records · Page 2Linked to original sources
PubMed1995

Sequence analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron-carrying arm of a redox loop.

Three genes, narH, narJ and narI, of the membrane-bound nitrate reductase operon of the denitrifying bacterium Thiosphaera pantotropha have been identified and sequenced. The derived gene products show high sequence similarity to the equivalent (beta, putative delta and gamma) subunits of the two membrane-bound nitrate reductases of the enteric bacterium Escherichia coli. All iron-sulphur cluster ligands proposed for the E. coli beta subunits are conserved in T. pantotropha NarH. Secondary structure analysis of NarJ suggests that this protein has a predominantly alpha-helical structure. Comparison of T. pantotropha NarI with the b-haem-binding integral membrane subunits of the E. coli enzymes allows assignment of His-53, His-63, His-186 and His-204 (T. pantotropha NarI numbering) as b-haem axial ligands and the construction of a three-dimensional model of this subunit. This model, in which the two b-haems are in different halves of the membrane bilayer, is consistent with a mechanism of energy conservation whereby electrons are moved from the periplasmic to the cytoplasmic side of the membrane via the haems. Similar movement of electrons is required in the membrane-bound uptake hydrogenases and membrane-bound formate dehydrogenases. We have identified two pairs of conserved histidine residues in the integral membrane subunits of these enzymes that are appropriately positioned to bind one haem towards each side of the membrane bilayer. One subunit of a hydrogenase complex involved in transfer of electrons across the cytoplasmic membrane of sulphate-reducing bacteria has structural resemblance to NarI.

Amino Acid Sequence↗
PubMed1994

Sequential assignments and identification of secondary structure elements of the colicin E9 immunity protein in solution by homonuclear and heteronuclear NMR.

1H-1H, 1H-15N, and 1H-1H-15N multidimensional NMR spectroscopic studies of the 86 amino acid protein that provides immunity against the DNase action of colicin E9 are reported. Through a combination of 2D NOESY and TOCSY and 3D TOCSY-HMQC, NOESY-HMQC, and HMQC-NOESY-HMQC experiments, almost complete 1H NMR and backbone 15N NMR assignments have been obtained, and the secondary structure of the protein has been partially elucidated. Approximately 50% of the protein forms three helices. The specificity determining region of the DNase immunity protein, identified from previously reported biochemical studies to include residues 32-40, is helical, indicating that the protein-protein interaction involves residues from at least one helix.

Amino Acid Sequence↗
PubMed1994

Characterization of the paramagnetic iron-containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase.

Electron paramagnetic resonance spectroscopy signals attributable to low-spin haem c in the oxidised protein and [4Fe-4S]1+ in the dithionite-reduced protein were identified, at low temperature, in Thiosphaera pantotropha periplasmic nitrate reductase. Spin integration of these signals as well as elemental analysis suggest a stoichiometry of 1.3-1.6 c-haem and 1 [4Fe-4S] cluster per enzyme molecule. The Em (at pH 7.4) of the [4Fe-4S]2+,1+ couple, -160 mV, means that it is unlikely to be physiologically reducible. Peptide sequences from the 90 kDa subunit indicate that the enzyme is a member of the family of molybdopterin guanine dinucleotide-binding polypeptides, the majority of which possess a putative [4Fe-4S] cluster binding sequence and thus may also bind a (low potential) iron-sulphur cluster.

Amino Acid Sequence↗
PubMed1994

Tandem overproduction and characterisation of the nuclease domain of colicin E9 and its cognate inhibitor protein Im9.

We report the overproduction of the non-specific endonuclease domain of the bacterial toxin colicin E9 and its preliminary characterisation in vitro. The enzymatic colicins (61 kDa) are normally released from producing cells in a complex with their cognate inhibitors, known as the immunity proteins (9.5 kDa). Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N-terminal and central regions of the toxin and a heterodimer comprising the catalytically active C-terminal domain of the colicin bound to its intact immunity protein, Im9. N-terminal amino acid sequencing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heterogeneous, thus making subsequent mechanistic and structural analysis difficult. This problem was circumvented by selectively overexpressing the C-terminal 15-kDa nuclease domain of colicin E9 in tandem with its cognate inhibitor in Escherichia coli. This tandem overexpression strategy allowed high-level production of a 25-kDa protein complex comprising the C-terminal DNase domain of colicin E9 tightly bound to its specific inhibitor Im9, thus masking the anticipated toxicity of the nuclease. The DNase domain was then separated from Im9 under denaturing conditions, refolded by removal of the denaturant and the renatured protein shown to possess both endonuclease and Im9 binding activity. These results describe a novel method for the overproduction of a nuclease in bacteria by co-expressing its specific inhibitor and lay the foundations for a full mechanistic, biophysical and structural characterization of the isolated DNase domain of the colicin E9 toxin.

Amino Acid Sequence↗
PubMed1994

Product-induced stabilization of tertiary and quaternary structure in Escherichia coli dehydroquinase.

The effects of irreversibly attaching product at the active site of type I dehydroquinase from Escherichia coli have been investigated at the level of the secondary, tertiary, and quaternary structure of the protein by spectroscopic and hydrodynamic techniques. The results agree with the previously identified stabilization of the enzyme but show for the first time that the protein dimer is also stabilized and suggest that the E. coli dehydroquinase subunit may be bilobal.

Circular Dichroism↗
PubMed1994

Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.

Cell Membrane↗
PubMed1994

Employment-based health benefits: analysis of the April 1993 Current Population Survey.

The April 1993 CPS differs from the March 1993 CPS in a number of respects. The April 1993 CPS supplement surveys only workers, whereas the March CPS examines the noncash benefits received by all Americans. The April CPS asks workers about health coverage in the week in which the questions were fielded, whereas the March CPS asks about coverage in the preceding year. In April 1993, there were 112.5 million civilian American workers between the ages of 18 and 64 with jobs. Eighty-two million (73 percent) of them worked for an employer that sponsored a health insurance plan, and 65 million (58 percent of all workers) participated in their employer's health plan. About one-third of workers at firms with fewer than 10 employees had employers who offer health benefits; about one-quarter of all of the workers in these firms participated in their employer's plan. Conversely, 94 percent of workers at firms with more than 1,000 employees had an employer who sponsored health benefits, and over 77 percent of these workers participated in their employer's plan. There are 16.5 million American workers whose employers sponsored health benefits but who did not participate in these benefits. Over one-half of these workers (8.5 million) chose not to be covered. Another 36 percent of these workers (5.9 million) did not participate because they were ineligible or denied coverage. Over 66 percent of the ineligible workers did not participate because they were part-time, contract, or temporary workers. Another 26 percent had not yet completed a probationary period. Among the reasons that those who chose not to participate in their employer's coverage, the vast majority (75 percent) stated they were covered by another health care plan. Twenty-nine percent stated that they chose not to purchase coverage because it was too costly or that they did not need or want the coverage. In 1993, there were 16.7 million workers with no health insurance coverage. The vast majority of these workers (95 percent) were employed by private employers. Sixty-six percent of the workers with no health insurance coverage were self-employed or worked for firms with fewer than 100 employees.

Adult↗
PubMed1994

Factors influencing prehospital delay in patients experiencing chest pain.

BACKGROUND: Patients experiencing chest pain (or other cardiac symptoms) in the community delay hours, even days, in seeking medical care. Given the potential effectiveness of thrombolytic therapy, it is important to determine patterns of patient delay. OBJECTIVES: A descriptive study was conducted to determine the amount of time patients delayed in seeking medical care when experiencing chest pain and the characteristics of patients delaying longer than 3 hours. METHODS: Adult patients (N = 77) admitted to an emergency department with chest pain participated. Data were obtained using the Response to Symptoms Questionnaire and chart review. RESULTS: The mean prehospital delay times (ie, symptom onset to hospital arrival) was 25.4 hours (+/- 46.5), and the median was 5 hours. Delay groups were divided into those arriving less than 3 hours (n = 31 or 40%) and those arriving more than 3 hours (n = 46 or 60%) after symptom onset. Three hours was chosen based on a mean time, taken from the literature, of 84 minutes spent in the emergency department so that patients in the first group would be able to receive thrombolysis within 4 hours. Univariate statistical analyses revealed that advanced age, symptom onset with a family member present, and self-determination of symptoms as not being serious in nature all contributed to a delay of longer than 3 hours. CONCLUSIONS: Critical care nurses and physicians should focus on patients at high risk for prolonged delay and educate the public about action to take if a family member experiences cardiac symptoms.

Adult↗
PubMed1993

Purification of hydroxylamine oxidase from Thiosphaera pantotropha. Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification.

Thiosphaera pantotropha, a Gram-negative heterotrophic nitrifying bacterium, expresses a soluble 20 kDa monomeric periplasmic hydroxylamine oxidase that differs markedly from the hydroxylamine oxidase found in autotrophic bacteria. This enzyme can use the periplasmic redox proteins, cytochrome c551 and pseudoazurin as electron acceptors, both of which can also donate electrons to denitrification enzymes. A model of electron transfer is proposed, that suggests a coupling of nitrification and provides a mechanism by which nitrification can play a role in dissipating reductant.

Electron Transport↗
PubMed1992

In vivo and in vitro characterization of overproduced colicin E9 immunity protein.

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.

Bacterial Proteins↗
PubMed1990

Excitatory amino acids toxicity in cerebellar granule cells in primary culture.

Primary cultures of cerebellar granule cells represent a valuable neuronal system for the study of the physiology, biochemistry and toxicology of excitatory amino acids. Using this system, we now report the characterization of the biochemical and toxicological action of a toxic mussels extract, containing the excitatory amino acid domoic acid. The results obtained using toxic mussels extract, non-toxic mussels extract and purified domoic acid, provide evidence for a synergistic action of domoic acid with other potentially neurotoxic excitatory amino acids, normally present in mussels.

Amino Acids↗
PubMed1989

Glomerulitis induced by cationized bovine serum albumin in the rat.

The effects of injected native and cationized bovine serum albumin (BSA- and BSA+ respectively) were evaluated in rats which subsequently received anti-BSA. Thrombocytopenia, low creatinine clearance (Ccr), increased proteinuria, capillary swelling, mild tuft necrosis and BSA+ deposits in glomeruli resulted within 24 h of BSA+ injection. Later BSA+ produced mesangial expansion glomerular capillary wall (GCW) thickening and deposits of BSA+ accompanied by rabbit anti-BSA and rat anti-BSA which correlated well with small mesangial, subendothelial and subepithelial electron-dense granular accumuli. These latter enlarged considerably after the injection of anti-BSA. BSA- controls showed minimal or no lesions. The disappearance from the blood (t1/2) of a single dose of immune complexes (IC) prepared with chromatography-purified, radioiodinated anti-BSA - BSA- and BSA+ was determined in another group of rats. The t1/2 of BSA- anti-BSA was 42.8 h (95% confidence: 39.8-46.2) while that of BSA+ anti-BSA was 52.5 h (48.1-57.8). These results suggested that serum sickness glomerulitis developed only in rats injected with BSA+, due to in situ IC which presumably grew by accretion of foreign anti-BSA. Circulating IC may have developed and colocated with the latter, with dissociation and recombination at these sites. It is postulated that the functional-immunomorphological changes and the slow removal of cationized IC reported herein could be explained by the highly positive net charge of the injected antigen.

Animals↗
PubMed1987

A single autoantigen in Goodpasture's syndrome identified by a monoclonal antibody to human glomerular basement membrane.

A mouse monoclonal antibody (P1) to the autoantigenic component of human glomerular basement membrane (GBM) was used to study the immunochemistry and tissue distribution of the Goodpasture antigen and the specificity of the human autoimmune response in Goodpasture's syndrome (anti-GBM disease). In solid phase assays, monoclonal antibody P1 bound to collagenase-solubilized human GBM (the ligand used in assays for human autoantibody), but not to other biochemically defined components of basement membrane. On Western blotting, P1 bound to the same 6 bands in solubilized GBM (between 26 and 58 kilodaltons with major bands at 26 and 54 kilodaltons) that were recognized by sera from all 42 patients studied with anti-GBM disease. Preincubation with sera from 8/8 patients blocked the subsequent binding of P1 from 83 to 89% on densitometer scanning of the Western blot; and preincubation with P1 blocked the binding of sera from 6/6 patients from 58 to 89%. Indirect immunofluorescence and immunoperoxidase studies revealed that the pattern of binding of P1 was identical to that of antibody eluted from the kidneys of a patient with Goodpasture's syndrome; there was linear binding to GBM, Bowman's capsule, and distal tubular basement membrane. In addition, P1 bound to basement membranes in lung and choroid plexus, and to membranes of the lens capsule, choroid, and retina of the eye and cochlea, but not to other organs studied. It is concluded that there is a single major autoantigenic component of human GBM (the Goodpasture antigen), which is present on fragments of different molecular weight in the collagenase digest. This antigen is distributed throughout well-defined basement membranes known to be involved in both Goodpasture's and Alport's syndromes. Human anti-GBM antibodies bind to the same (or closely related) determinants which are recognized by P1, demonstrating that the autoimmune response in Goodpasture's syndrome is of highly restricted specificity.

Anti-Glomerular Basement Membrane Disease↗
PubMed1986

Comparison of neurothermography and contrast myelography.

Eighty consecutive patients who first had an electronic infrared thermogram (neurothermogram) and a subsequent oil myelogram were studied retrospectively. All patients had cervical and/or lumbosacral radiculopathy for 90 days. Negative neurothermograms were predictive of negative myelograms in 93% of our series. Positive neurothermograms were predictive of positive myelograms in 71% of the patients. The neurothermogram is not a specific test for intraspinal axis lesions as a cause of radiculopathy. Proximal neuropathic lesions of other causes will also produce abnormalities of the sympathetic autonomic (peripheral) nervous system. The major pathophysiologic findings detected by neurothermograms are complimentary to the EMG, NCVS, and late response techniques. Neurothermography was successful in predicting the outcome of myelography in 82% of patients in this study.

Adult↗
PubMed1986

General practitioners' attitudes towards the limited list.

From a one in 10 randomly selected sample of Scottish general practitioners 260 (85%) responded to a series of attitude statements concerned with limitation of NHS prescribing which was introduced in April 1985. Although a majority of respondents were against the current limited list scheme, two-fifths were in favour of it and many of the objectors were in favour of the principle of limitation but not the way in which it had been implemented.

Attitude of Health Personnel↗