Search PubMed⌕ Search

Biomedical subjects

C Anthony

Publications and source records attributed to C Anthony.

At least 37 records · Page 2Linked to original sources
PubMed1998

A life review interview guide: a structured systems approach to information gathering.

This clinical project introduces a guide to provide the interviewer with a methodological systems approach for information gathering in the life review process. The guide is structured in such a way as to elicit positive life events for the purpose of enhancing a sense of well-being as well as to elicit negative life events to encourage the client to address unresolved loss-grief issues. In effect, the Life Review Interview Guide serves to promote high self-esteem and to assist the interviewee through the grieving process. In addition, the Guide assisted the student-interviewer in formulating and selecting a wide range of questions.

Adult↗
PubMed1997

The methanol oxidation genes mxaFJGIR (S) ACKLD in Methylobacterium extorquens.

MxaJ is a protein of unknown function encoded by mxaJ in the mxaFJGI operon. We have constructed a mxaJ mutant of M. extorquens with a deletion which does not affect transcription of downstream genes. It contained cytochrome cL (MxaG), but neither subunit of methanol dehydrogenase (MxaF and MxaI). MxaJ is probably involved in processing this enzyme. We have sequenced the region between mxaFJGI and five other methanol oxidation genes, mxaACKLD; it includes one open reading frame (mxaR) and a possible second open reading frame (mxaS), demonstrating the presence in M. extorquens of the following gene cluster: mxaFJGIR(S) ACKLD.

Alcohol Oxidoreductases↗
PubMed1996

Quinoprotein-catalysed reactions.

This review is concerned with the structure and function of the quinoprotein enzymes, sometimes called quinoenzymes. These have prosthetic groups containing quinones, the name thus being analogous to the flavoproteins containing flavin prosthetic groups. Pyrrolo-quinoline quinone (PQQ) is non-covalently attached, whereas tryptophan tryptophylquinone (TTQ), topaquinone (TPQ) and lysine tyrosylquinone (LTQ) are derived from amino acid residues in the backbone of the enzymes. The mechanisms of the quinoproteins are reviewed and related to their recently determined three-dimensional structures. As expected, the quinone structures in the prosthetic groups play important roles in the mechanisms. A second common feature is the presence of a catalytic base (aspartate) at the active site which initiates the reactions by abstracting a proton from the substrate, and it is likely to be involved in multiple reactions in the mechanism. A third common feature of these enzymes is that the first part of the reaction produces a reduced prosthetic group; this part of the mechanism is fairly well understood. This is followed by an oxidative phase involving electron transfer reactions which remain poorly understood. In both types of dehydrogenase (containing PQQ and TTQ), electrons must pass from the reduced prosthetic group to redox centres in a second recipient protein (or protein domain), whereas in amine oxidases (containing TPQ or LTQ), electrons must be transferred to molecular oxygen by way of a redox-active copper ion in the protein.

Alcohol Oxidoreductases↗
PubMed1996

Reconstitution of the quinoprotein methanol dehydrogenase from inactive Ca(2+)-free enzyme with Ca2+, Sr2+ or Ba2+.

The reconstitution of active holoenzyme containing calcium from inactive calcium-free methanol dehydrogenase, isolated from a moxA mutant of Methylobacterium extorquens, has a pH optimum of about pH 10, with a well defined pK for the process at pH 9.3. Two Ca2+ ions were irreversibly incorporated per alpha 2 beta 2 tetramer. Calcium could be replaced in the incorporation process by strontium or barium, the affinities for these ions being similar to that for Ca2+. Arrhenius plots for measurement of the activation energy of reconstitution were biphasic; the lower activation energy was typical of most biological processes, while the higher activation energy was at least three times greater, implying the involvement of a large conformational change during incorporation of the cations. The activation energy for incorporation of Ba2+ was considerably higher than that for incorporation of Ca2+. The novel disulphide bridge that is at the active site of the enzyme was not involved in the incorporation process. Studies of the time courses for incorporation of 45Ca2+, production of active enzyme and changes in absorption spectra failed to show any intermediates in the incorporation process.

Alcohol Oxidoreductases↗
PubMed1996

Characterization of a novel methanol dehydrogenase containing a Ba2+ ion at the active site.

The quinoprotein methanol dehydrogenase (MDH) contains a Ca2+ ion at the active site. Ca(2-)-free enzyme (from a processing mutant) was used to obtain enzyme containing Sr2+ or Ba2+, the Ba(2+)-MDH being the first enzyme to be described in which a Ba2+ ion functions at the active site. The activation energy for oxidation of methanol by Ba(2+)-MDH is less than half that of the reaction catalysed by Ca(2+)-MDH (a difference of 21.4 kJ/mol), and the Vmax value is 2-fold higher. The affinities of Ba(2+)-MDH for substrate and activator are very much less than those of Ca(2+)-MDH; the Km for methanol is 3.5 mM (compared with 3 microM) and the KA for ammonia is 52 mM (compared with 2 mM). The different activity of Ba(2+)-MDH is probably due to a change in the conformation of the active site, leading to a decrease in the free energy of substrate binding and hence a decrease in activation energy. The kinetic model for Ba(2+)-MDH with respect to substrate and activator is consistent with previous models for Ca(2+)-MDH. The pronounced deuterium isotope effect (6.0-7.6) is influenced by ammonia, and is consistent with activation of the pyrroloquinoline quinone reduction step by ammonia. Because of its low affinity for substrates, it is possible to prepare the oxidized form of Ba(2+)-MDH. No spectral intermediates could be detected during reduction by added substrate, and so it is not possible to distinguish between those mechanisms involving covalent substrate addition and those involving only hydride transfer.

Alcohol Oxidoreductases↗
PubMed1995

Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of methanol dehydrogenase from Methylobacterium extorquens.

The structure of methanol dehydrogenase (MDH) at 0.194 nm (1.94 A) has been used to provide a model structure for part of a membrane quinoprotein glucose dehydrogenase (GDH). The basic superbarrel structure is retained, along with the tryptophan-docking motifs. The active-site regions are similar, but there are important differences, the most important being that GDH lacks the novel disulphide ring structure formed from adjacent cysteines in MDH; in GDH the equivalent region is occupied by His-262. Because of the overall similarities in the active-site region, the mechanism of action of GDH is likely to be similar to that of MDH. The differences in co-ordination to the cation and bonding to the pyrrolo-quinoline quinone (PQQ) in the active site may explain the relative ease of dissociation of the prosthetic group from the holo-GDH. There are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site, the configuration of which influences substrate specificity. The proposed model is consistent in many respects with previous proposals for the active-site structure based on the effects of chemical modification on binding of PQQ and enzymic activity.

Alcohol Oxidoreductases↗
PubMed1995

The interaction of methanol dehydrogenase and its cytochrome electron acceptor.

A fluorescence method is described for direct measurement of the interaction between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. This has permitted a distinction to be made between factors affecting electron transfer and those affecting the initial binding or docking process. It was confirmed that the initial interaction is electrostatic, but previous conclusions with respect to the mechanism of EDTA inhibition have been modified. It is proposed that the initial 'docking' of MDH and cytochrome cL is by way of ionic interactions between lysyl residues on its surface and carboxylate groups on the surface of cytochrome cL. This interaction is not inhibited by EDTA, which we suggest acts by binding to nearby lysyl residues, thus preventing movement of the 'docked' cytochrome to its optimal position for electron transfer, which probably involves interaction with the hydrophobic funnel in the surface of MDH.

Alcohol Oxidoreductases↗
PubMed1995

The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens.

The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase. The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.

Acetobacter↗
PubMed1995

The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens.

All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation. One of these is between adjacent cysteine residues which form a novel ring structure in the active site. It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL. The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ). The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge. No free thiols were detected during the reaction cycle with cytochrome cL. Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme. Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site. It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction. The disulphide ring has no special function in the process of Ca2+ incorporation into the active site. It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.

Alcohol Oxidoreductases↗
PubMed1995

The refined structure of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens at 1.94 A.

BACKGROUND: Methanol dehydrogenase (MDH) is a bacterial periplasmic quinoprotein; it has pyrrolo-quinoline quinone (PQQ) as its prosthetic group, requires Ca2+ for activity and uses cytochrome cL as its electron acceptor. Low-resolution structures of MDH have already been determined. RESULTS: The structure of the alpha 2 beta 2 tetramer of MDH from Methylobacterium extorquens has now been determined at 1.94 A with an R-factor of 19.85%. CONCLUSIONS: The alpha-subunit of MDH has an eight-fold radial symmetry, with its eight beta-sheets stabilized by a novel tryptophan docking motif. The PQQ in the active site is held in place by a coplanar tryptophan and by a novel disulphide ring formed between adjacent cysteines which are bonded by an unusual non-planar trans peptide bond. One of the carbonyl oxygens of PQQ is bonded to the Ca2+, probably facilitating attack on the substrate, and the other carbonyl oxygen is out of the plane of the ring, confirming the presence of the predicted free-radical semiquinone form of the prosthetic group.

Alcohol Oxidoreductases↗
PubMed1995

A randomised double-blind comparison of intravenous pamidronate and clodronate in the hypercalcaemia of malignancy.

In conjunction with rehydration, the bisphosphonates are the treatment of choice for hypercalcaemia of malignancy. Single infusions of either pamidronate or clodronate are usually effective, but a direct comparison of the two agents given at the highest doses commonly used has not been performed. Forty-one patients (15 breast, 12 squamous carcinomas, four lymphomas, four bladder, two prostate and four others) with hypercalcaemia of malignancy (corrected serum calcium > 2.7 mmol l-1) persisting after 48 h of saline rehydration were randomly allocated to receive a 4 h intravenous (i.v.) infusion of either pamidronate 90 mg or clodronate 1500 mg. No other systemic anti-cancer treatment was prescribed. There were no significant differences in the post-hydration serum calcium values (mean 3.17 mmol l-1 for pamidronate and 3.06 mmol l-1 for clodronate), tumour type or frequency of bone metastases between the two treatments. One patient on each treatment died within 2 days and was not assessable for response. A total of 19/19 (100%) patients achieved normocalcaemia following pamidronate and 16/20 (80%) with clodronate. The median time to achieve normocalcaemia was 4 days (range 2-14) for pamidronate and 3 days (range 2-6) with clodronate. The median duration of normocalcaemia was 28 days (range 10-28+ days) after pamidronate and 14 days after clodronate (range 7-21 days) (P < 0.01). Two patients who failed to respond to clodronate were successfully treated with pamidronate and achieved normocalcaemia for 14 and > 28 days respectively. Two patients experienced fever after pamidronate but no significant toxicity was observed with either treatment. We conclude that both agents are effective in the management of hypercalcaemia of malignancy. At the doses studied, the effects of pamidronate are more complete and longer lasting than those of clodronate.

Bone Neoplasms↗
PubMed1994

New unified nomenclature for genes involved in the oxidation of methanol in gram-negative bacteria.

The system involving the oxidation of methanol to formaldehyde in Gram-negative methylotrophic bacteria is complex. A total of 32 genes have been reported, termed mox, for methanol oxidation, and it is possible that more will be identified. Some mox genes carrying out completely different functions have been given the same designations by different laboratories and others have been given separate designations that were later discovered to be the same. It is now important to change the mox nomenclature to remedy this confusing situation. This communication proposes a new nomenclature for genes involved in methanol oxidation based on currently known linkage groups.

Genes, Bacterial↗
PubMed1994

X-ray structure of PQQ-dependent methanol dehydrogenase.

The three-dimensional structure of the PQQ-dependent quinoprotein, methanol dehydrogenase from Methylobacterium extorquens AM1, has been determined at 3A resolution. The a2b2 tetrameric enzyme has a large a-chain of almost spherical form with a chain fold in which eight 4-stranded antiparallel b-sheets segments are arranged radially around a pseudo 8-fold molecular symmetry axis. The much smaller b-chain is surprisingly not globular, but has an extended conformation running across the surface of the alpha-subunit. The PQQ prosthetic group is buried within the large a-subunit located on the pseudo 8-fold molecular symmetry axis. It is surrounded by protein side-chains but not covalently bound. Associated with the PQQ are two unexpected features: a vicinal disulphide bridge formed between Cys103 and Cys104, and a calcium ion bound between the protein and the PQQ. Vicinal disulphide bridges forming highly distorted structures containing a cis peptide bond, have been proposed to be present in one or two enzymes but have not previously been available for detailed structural investigation. Activity studies have indicated that the ability of the enzyme to transfer electrons derived from the reduction of the alcohols to the specific cytochrome CL receptor is lost when the vicinal disulphide bridge is reduced. The roles of the calcium ions and the b-chain in the enzyme's activity remain to be determined.

Alcohol Oxidoreductases↗
PubMed1994

High-dose intravenous pamidronate for metastatic bone pain.

The bisphosphonates are able to relieve pain from metastatic bone disease and, when given intravenously, may promote bone healing of lytic metastases. In this study, the aim was to assess the acute effects of a single 'high-dose' intravenous treatment with pamidronate on pain, mobility, analgesic consumption and quality of life (QOL). Thirty-four normocalcaemic patients with painful progressing bone metastases (22 from breast, five prostate and seven others) received a single intravenous infusion of 120 mg of pamidronate as palliative therapy. No other systemic therapy or drugs known to influence bone metabolism were administered during the study. Patients' subjective response to treatment was assessed weekly with a pain questionnaire recording a composite of pain intensity, mobility, performance status and analgesic consumption. In addition, patients completed the Rotterdam Symptom Check List (RSCL) for measurement of QOL and a mobility questionnaire. The mean reduction in the pain questionnaire score (recorded on at least two occasions) was 25% [standard error (s.e.) 3%, range 0-75%]. Twenty patients (59%) showed a > or = 20% improvement and were classified as responders. The median duration of symptomatic response was 12 (range 4-24 +) weeks. The responding patients showed a reduction in RSCL score (improvement in QOL) from 35% before treatment to 27% at 6 weeks, but no significant improvement was noted in non-responders. Twenty-one patients were retreated with pamidronate when their symptoms deteriorated again. Eight out of 15 responders showed a second reduction in pain score of > or = 20%, but this was not seen in any of the six non-responders. Five patients have remained well with no additional treatment for their disease other than repeat infusions of pamidronate every 3-6 months. Treatment was well tolerated. Eight (24%) experienced fever after the first treatment only, and four had asymptomatic, biochemical evidence of hypocalcaemia. The acute inhibition of osteoclastic bone resorption induced by a single high-dose treatment with pamidronate can provide useful palliation for patients with bone metastases. Responding patients may be retreated as symptoms dictate to good effect. We are currently running a phase III double-blind trial with high-dose pamidronate for progressive painful metastatic bone disease to exclude any placebo effect and observer bias.

Bone Neoplasms↗
PubMed1994

The active site of methanol dehydrogenase contains a disulphide bridge between adjacent cysteine residues.

Adjacent cysteine residues can only form disulphide bridges in a distorted structure containing a cis-peptide link. Such bridges are extremely uncommon, identified so far in the acetyl choline receptor alone where the structure of the bridge is undetermined. Here we present the first molecular description of a disulphide bridge of this type in the quinoprotein methanol dehydrogenase from Methylobacterium extorquens. We show that this structure occurs in close proximity to the pyrrolo-quinoline quinone prosthetic group and a calcium ion in the active site of the enzyme. This unusual disulphide bridge appears to play a role in the electron transfer reaction mediated by methanol dehydrogenase.

Alcohol Oxidoreductases↗