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A J Thomson

Publications and source records attributed to A J Thomson.

At least 55 records · Page 3Linked to original sources
PubMed1999

Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy.

The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.

Azides↗
PubMed1999

Vaccine protocols to optimise the protective efficacy of BCG.

SETTING: A deer model has been developed to study protection produced with BCG vaccination, against infection and the development of pathology, following experimental intratonsilar infection with virulent Mycobacterium bovis. OBJECTIVE: To determine how the dose of vaccine, the route of vaccination, the viability of the vaccine and exposure to glucocorticoids at the time of vaccination, may affect the protective efficacy of BCG vaccines. DESIGN: Deer were vaccinated with BCG and later challenged with virulent M. bovis via the tonsilar route. Protection against infection and development of disease was evaluated at necropsy six months after challenge with M. bovis, by histological examination and microbial culture. RESULTS: Significant protection against infection and disease were obtained following boosting with two low doses (5 x 10(4) cfu) or moderate doses (5 x 10(7) cfu) of live (freshly cultured and lyophilized) BCG. Inferior levels of protection were obtained with high dose (5 x 10(8) cfu) of live BCG. Similar levels of protection were found with vaccines given subcutaneously or via the tonsilar route. Killed vaccine in a mineral-oil adjuvant did not evoke protective immunity and treatment with dexamethasone prior to vaccination with live BCG ablated its efficacy. Protection against infection did not correlate with skin test delayed type hypersensitivity (DTH) or lymphocyte transformation to tuberculin. CONCLUSIONS: Two doses of live BCG gave significant protection against experimental infection and disease caused by virulent M. bovis. Single dose vaccine protected against disease but not infection. Vaccines administered at a dosage which did not evoke DTH, provided protection against tuberculosis infection and disease.

Adjuvants, Immunologic↗
PubMed1999

Expression of intercellular adhesion molecules ICAM-1 and ICAM-2 in human endometrium: regulation by interferon-gamma.

The purpose of this study was to localize intercellular adhesion molecule (ICAM)-1 and ICAM-2 in human endometrium and myometrium throughout the menstrual cycle, and to determine whether the expression of these molecules is regulated by interferon (IFN)-gamma. ICAM-1 and ICAM-2 distribution was examined in endometrial biopsies by immunocytochemistry, and Northern blotting was used to quantify ICAM-1 and ICAM-2 mRNA expression in isolated endometrial glands. Stromal fibroblast cultures were exposed to IFN-gamma and the effect on expression of ICAM-1 and ICAM-2 was determined by immunocytochemistry and Northern blotting. ICAM-1 was localized in vivo to the apical surface of the glandular epithelium, the vascular endothelium and endometrial stromal cells throughout the menstrual cycle. Stromal expression of ICAM-1 was up-regulated in menstrual specimens. Northern blotting confirmed the presence of ICAM-1 mRNA in isolated endometrial glands. The expression of ICAM-1 antigen and message was increased in stromal cell culture after incubation with IFN-gamma in a time-dependent manner, suggesting that this cytokine stimulates the expression of ICAM-1 in the endometrial stroma. ICAM-2 antigen expression was restricted to the vascular endothelium. ICAM-2 mRNA was absent in endometrial glands. The widespread distribution of ICAM-1 in human endometrium suggests that this molecule is involved in the process of menstruation, the functioning of glands, blood vessels and stroma, and in regulating leukocyte trafficking into the tissue. ICAM-2 is restricted to the vascular endothelium where it might modulate leukocyte invasion of the stroma and myometrial connective tissue.

Antigens, CD↗
PubMed1999

Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process.

Inflammatory mediators in the cervix, placenta and fetal membranes play a crucial role in human parturition. The aim of this study was to determine whether the upper and lower segments of the myometrium are infiltrated by inflammatory cells during pregnancy and parturition. Myometrial biopsies were obtained from non-pregnant women, and pregnant women at term before and after the onset of spontaneous labour. Subpopulations of inflammatory cells were identified using immunocytochemistry. The intercellular adhesion molecules, 1 and 2, platelet endothelial cell adhesion molecule, vascular cell adhesion molecule and E-selectin were immunolocalized to investigate their involvement in leukocyte accumulation. Histological analysis demonstrated that inflammatory cells, predominantly neutrophils and macrophages, infiltrate human myometrium during spontaneous labour at term. The infiltrate is predominant in the lower uterine segment but is also present in the upper segment. Increased expression of E-selectin was found on the vascular endothelium of biopsies obtained during labour, suggesting a role for this molecule in the accumulation of leukocytes. These results suggest that inflammatory cell infiltration is part of the physiological mechanisms that occur in the myometrium during parturition. Further understanding of this process may suggest new strategies aimed at preventing preterm delivery.

Cell Adhesion Molecules↗
PubMed1998

Spectroscopic characterization of a novel multiheme c-type cytochrome widely implicated in bacterial electron transport.

NapC is a member of a family of bacterial membrane-anchored tetra-heme c-type cytochromes that participate in a number of respiratory electron transport pathways. They are postulated to mediate electron transfer between membrane quinols/quinones and soluble periplasmic enzymes. The water-soluble heme domain of NapC has been expressed as a periplasmic protein. Mediated redox potentiometry and characterization by UV-visible, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies demonstrates that soluble NapC contains four low spin hemes, each with bis-histidine axial ligation and with midpoint reduction potentials of -56, -181, -207, and -235 mV.

Amino Acid Sequence↗
PubMed1998

Voltammetric studies of the reactions of iron-sulphur clusters ([3Fe-4S] or [M3Fe-4S]) formed in Pyrococcus furiosus ferredoxin.

Reactions of the [3Fe-4S] cluster and various metallated [M3Fe-4S] adducts co-ordinated in the ferredoxin from the hyperthermophile Pyrococcus furiosus have been studied by protein-film voltammetry, bulk-solution voltammetry, solution kinetics and magnetic CD (MCD). The [3Fe-4S] cluster exhibits two couples, [3Fe-4S]+/0 and [3Fe-4S]0/2-. Film voltammetry is possible over a wide pH range (2-8), revealing that the [3Fe-4S]+/0 couple shows a complex pH dependence with pKred1=2.8, pKox=4.9 and pKred2=6.7. From MCD, pKred1 corresponds with protonation of [3Fe-4S]0 to give a spectroscopically distinct species, as reported for ferredoxins from Azotobacter and Sulfolobus. The status of the disulphide/disulphydryl entity makes no significant difference to the data (given for the -S-S- form). Formation of the hyper-reduced [3Fe-4S]2- state is observed, requiring 3H+ for the overall 3e- reduction of [3Fe-4S]+, the change therefore being electroneutral. By comparison with the ferredoxin from Desulfovibrio africanus, uptake of Fe(II) and other M(II) by [3Fe-4S]0 to give [M3Fe-4S] clusters is slow (t1/2>10 min at room temperature, slower still if the protein is adsorbed on the electrode), whereas reaction with Tl(I) to produce [Tl3Fe-4S] is very rapid (t1/2<<1 s), suggesting that co-ordination of Tl does not require reorganization of the protein structure. Rates of formation of [3Fe-4S] from [M3Fe-4S] adducts increase sharply at high potentials, showing that metal release involves a labile 'super-oxidized' [M3Fe-4S]3+ state.

Bacterial Proteins↗
PubMed1998

Randomised trial of nitric oxide donor versus prostaglandin for cervical ripening before first-trimester termination of pregnancy.

BACKGROUND: Vaginal administration of the nitric oxide donor isosorbide mononitrate can induce effective ripening of the human cervix. We investigated whether this drug is associated with fewer side-effects than prostaglandins when used to ripen the cervix before first-trimester surgical termination of pregnancy, and assessed whether the extent of cervical ripening it induces is clinically sufficient. METHODS: 66 primigravid women scheduled for surgical termination were assigned to receive before surgery, per vaginam, isosorbide mononitrate 40 mg or 80 mg, or the prostaglandin analogue gemeprost 1 mg. The primary measured outcome was onset of new symptoms before termination of pregnancy. FINDINGS: More women remained symptom-free after isosorbide mononitrate than after gemeprost (28/44 [64%] vs 3/22 [14%], p<0.005). Pretreatment with gemeprost resulted in abdominal pain in 73% of women and vaginal bleeding in 32% compared with 3% and 0%, respectively, after isosorbide mononitrate, whereas, more women developed headache after isosorbide mononitrate (27%) than after gemeprost (0%). Cervical resistance and measured intraoperative blood loss were lowest after pretreatment with gemeprost. The measured cervical resistance and intraoperative blood loss with either dose of isosorbide mononitrate did not differ from those in a comparison group of 22 parous women not in the randomised trial. INTERPRETATION: Pretreatment with isosorbide mononitrate to ripen the cervix before first-trimester termination of pregnancy is associated with fewer side-effects than gemeprost treatment and adequately decreases cervical resistance. Isosorbide mononitrate could be used as an alternative to gemeprost for this indication.

Abortifacient Agents, Nonsteroidal↗
PubMed1998

CuA and CuZ are variants of the electron transfer center in nitrous oxide reductase.

Nitrous oxide reductase (N2OR) is a dimeric copper-dependent bacterial enzyme that catalyzes the reduction of N2O to N2 as part of the denitrification pathway. In the absence of an x-ray crystal structure, the current model of the nature of the copper sites within the enzyme is based on four copper atoms per monomer and assigns two copper atoms to an electron transfer center, CuA, a bis-thiolate-bridged dinuclear copper center found to date only in N2OR and cytochrome c oxidase, and two copper atoms to a second dinuclear center, CuZ, presumed to be the site of catalysis. Based on detailed analysis of the low temperature magnetic CD spectra of N2OR, this paper revises the current model and proposes that both CuA and CuZ are variants of an electron transfer center and hence that all of the observed optical features are due to this electron transfer center. It is proposed further that the presence of these different forms provides a mechanism for the delivery of two electrons to an active site comprising copper ions lacking thiolate coordination.

Amino Acid Sequence↗
PubMed1998

The reaction of halides with pulsed cytochrome bo from Escherichia coli.

Cytochrome bo forms complexes with chloride, bromide and iodide in which haem o remains high-spin and in which the '630 nm' charge-transfer band is red-shifted by 7-8 nm. The chloride and bromide complexes each have a characteristic set of integer-spin EPR signals arising from spin coupling between haem o and CuB. The rate and extent of chloride binding decreases as the pH increases from 5.5 to 8.5. At pH 5.5 the dissociation constant for chloride is 2 mM and the first-order rate constant for dissociation is 2 x 10(-4) s-1. The order of rate of binding, and of affinity, at pH 5.5 is chloride (1) > bromide (0.3) >iodide (0.1). It is suggested that the halides bind in the binuclear site but, unlike fluoride, they are not direct ligands of the iron of haem o. In addition, both the stability of the halide complexes and the rate of halide binding seem to be increased by the co-binding of a proton.

Binding Sites↗
PubMed1998

The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases.

EPR spectra at liquid helium temperatures and MCD spectra at room temperature and 4.2 K are presented for fully oxidized nitric oxide reductase (NOR) from Pseudomonas stutzeri. The MCD spectra show that the enzyme contains three heme groups at equivalent concentrations but distinctive in their axial coordination. Two, in the low-spin ferric state at all temperatures, give rise to infrared charge-transfer transitions which show the hemes to have bis-histidine and histidine-methionine ligation, respectively. The EPR spectra show them to be magnetically isolated. The third heme has an unusual temperature-dependent spin state and spectroscopic features which are consistent with histidine-hydroxide coordination. No EPR signals have been detected from this heme. Together with its unusual near-infrared MCD, this suggests a spin-spin interaction between this heme and another paramagnet. The three hemes account for only 75% of the iron content, and it is concluded that the additional paramagnet is a mononuclear ferric ion. These results provide further evidence that NOR is indeed structurally related to heme-copper oxidases and that it contains a heme/non-heme iron spin-coupled pair at the active site.

Binding Sites↗
PubMed1998

Farmed deer: a large animal model for stress.

In the early classic period it was suggested by Heracleitus that a static unchanged state was not the natural condition, but rather that the capacity to undergo constant change was intrinsic to all living things. After that, Empedoeles, circa 500 BC, proposed that for living organisms to survive they require a harmonious mixture of elements. Hippocrates, around 400 BC, developed this concept further by suggesting that health is a state of harmonious balance of these elements, equating disease to a state of disharmony and introducing the concept that nature heals disease (Noysvn Fyseiw/atrui). Soon after, Epicurus extended these ideas to include among these healing forces, the mind, writing that imperturbability of mind is desirable. Claude Bernard introduced the concept of the milieu interieur or the internal physiologic environment, describing its constancy as essential in an external environment typified by its variability. Walter Cannon described Bernard's constancy as Homeostasis and identified the "fight or flight reflex," linking the adaptive response and catecholamine secretion, with extreme levels of activation-producing pathology. Selye outlined four stages of the stress response: 1) the "alarm reaction" characterized by an immediate activation of the sympathetic-adrenomedullary axis (SA); 2) a "resistance phase" characterized by hypothalamic pituitary-adrenal axis (HPA) activation; 3) a stage of adrenal hypertrophy, gastrointestinal ulceration, along with thymic and lymphoid atrophy; and 4) an exhaustion phase and finally death (1).

Animal Husbandry↗
PubMed1998

The dinuclear center of cytochrome bo3 from Escherichia coli.

For the study of the dinuclear center of heme-copper oxidases cytochrome bo3 from Escherichia coli offers several advantages over the extensively characterized bovine cytochrome c oxidase. The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies. Cytochrome bo3 is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in CcO. The absence of CuA and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o3. Careful analysis allows us to distinguish between small molecules that bind to heme o3, those which are ligands of CuB, and those which react to yield higher oxidation states of heme o3. Here we review results from our studies of the reactions of fast cytochrome bo3 with formate, fluoride, chloride, azide, cyanide, NO, and H2O2.

Animals↗
PubMed1998

Ultrasound detection of vault haematoma following vaginal hysterectomy.

OBJECTIVE: To assess whether ultrasound detection of vault haematoma can be used as a predictor of post-operative morbidity following vaginal hysterectomy. DESIGN: Prospective observational study of consecutive cases of vaginal hysterectomy performed between 1991 and 1994. SAMPLE: Two hundred and twenty-three women having undergone vaginal hysterectomy. METHODS: All women had transvaginal ultrasound examination by an independent observer on the third post-operative day. Routine observations and clinical assessments were made by established management protocol, by staff blinded to the ultrasound findings. MAIN OUTCOME MEASURES: Febrile morbidity; haemoglobin drop; need for blood transfusion; length of stay in hospital; re-admission rate and length of stay. RESULTS: Of the 223 scanned women, 55 (25%) had a vault haematoma. This group was compared with the 168 women with no haematoma. Significant increases in febrile morbidity (31% vs 7%), post-operative haemoglobin drop (2.5 g/dL vs 1.6 g/dL), need for blood transfusion (14.5% vs 1.2%), representation to hospital (25.5% vs 5.4%) and length of hospital stay (8.87 days vs 6.25 days) were seen in the haematoma group. CONCLUSION: Ultrasound detection of vault haematoma following vaginal hysterectomy is a common finding associated with increased febrile morbidity, need for blood transfusion, longer hospital stay and higher re-admission rate. In view of increasing demand for early discharge, driven by purchasers and patients, post-operative ultrasound identifies a high risk population and is both convenient and noninvasive. To further reduce morbidity, it also offers the opportunity to perform a controlled trial of prophylactic antibiotics in this identified subset.

Female↗
PubMed1997

Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB.

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.

Bacterial Proteins↗