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Biomedical subjects

A M Edwards

Publications and source records attributed to A M Edwards.

At least 55 records · Page 3Linked to original sources
PubMed2000

NMR structure determination and structure-based functional characterization of conserved hypothetical protein MTH1175 from Methanobacterium thermoautotrophicum.

The solution structure of MTH1175, a 124-residue protein from the archaeon Methanobacterium thermoautotrophicum has been determined by NMR spectroscopy. MTH1175 is part of a family of conserved hypothetical proteins (COG1433) with unknown functions which contains multiple paralogs from all complete archaeal genomes and the archaeal gene-rich bacterium Thermotoga maritima. Sequence similarity indicates this protein family may be related to the nitrogen fixation proteins NifB and NifX. MTH1175 adopts an alpha/beta topology with a single mixed beta-sheet, and contains two flexible loops and an unstructured C-terminal tail. The fold resembles that of Ribonuclease H and similar proteins, but differs from these in several respects, and is not likely to have a nuclease activity.

Amino Acid Sequence↗
PubMed2000

Protein production: feeding the crystallographers and NMR spectroscopists.

Protein purification efforts for structural genomics will focus on automation for the readily-expressed proteins, and process development for the more difficult ones, such as membrane proteins. Thousands of proteins are expected to be produced in the next few years. The purified proteins will be valuable reagents for the entire research community.

Animals↗
PubMed2000

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, approximately 20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Cloning, Molecular↗
PubMed2000

Riboflavin photodegradation and photosensitizing effects are highly dependent on oxygen and ascorbate concentrations.

Riboflavin (RF) is a normal component of the eye lens which triggers a strong photosensitizing activity when exposed to light. Upon irradiation with short wavelength radiations below 400 nm, RF-photosensitized damage may occur. However, vitamin C is present at high concentrations in the normal lens and plays an important role in inhibiting these photosensitization processes. An in vitro simple model was used with the objective of understanding better the relationships between vitamin C and oxygen concentrations on the mechanisms of RF-mediated photodegradation of tryptophan (Trp), a target particularly sensitive to photo-oxidation. Under nitrogen, the RF decomposition reached its maximal value, and vitamin C and Trp photo-oxidation was negligible. When increasing oxygen pressure, RF photodegradation dropped and vitamin C photo-oxidation strongly increased and was maximal at 100% O2. RF-induced photodegradation of Trp first increased with oxygen concentration, up to 40 microM O2, and then decreased. RF and Trp degradation were significantly protected by vitamin C so that no more than 20% of the substrates concentration were oxidized in the presence of vitamin C higher than 0.8 mM. From our results we conclude that in the specific conditions of the normal lens, the high vitamin C concentration (2 mM) is compatible with the UVA radiation hazard, despite the presence of RF. However, if lenticular vitamin C decreases below 0.8 mM, photodegradation of RF may occur and Trp may therefore be photo-oxidized by a Type-I mechanism.

Ascorbic Acid↗
PubMed1999

The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding.

Replication protein A (RPA), the eukaryote single-stranded DNA-binding protein (SSB), is a heterotrimer. The largest subunit, RPA70, which harbours the major DNA-binding activity, has two DNA-binding domains that each adopt an OB-fold. The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA-binding activity but the mechanism of DNA binding is unknown. We have determined the crystal structure of the proteolytic core of RPA32 and RPA14, which consists of the central two-thirds of RPA32 and the entire RPA14 subunit. The structure revealed that RPA14 and the central part of RPA32 are structural homologues. Each subunit contains a central OB-fold domain, which also resembles the DNA-binding domains in RPA70; an N-terminal extension that interacts with the central OB-fold domain; and a C-terminal helix that mediate heterodimerization via a helix-helix interaction. The OB-fold of RPA32, but not RPA14, possesses additional similarity to the RPA70 DNA-binding domains, supporting a DNA-binding role for RPA32. The discovery of a third and fourth OB-fold in RPA suggests that the quaternary structure of SSBs, which in Bacteria and Archaea are also tetramers of OB-folds, is conserved in evolution. The structure also suggests a mechanism for RPA trimer formation.

Amino Acid Sequence↗
PubMed1999

Binding of elongin A or a von Hippel-Lindau peptide stabilizes the structure of yeast elongin C.

Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL. To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region. Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers. Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer. Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins. These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL.

Amino Acid Sequence↗
PubMed1999

Visible light-induced photooxidation of glucose sensitized by riboflavin.

We conducted this study to evaluate the oxidation of glucose induced by visible light in the presence of sensitizers such as methylene blue and flavins (i.e., flavin mononucleotide and riboflavin). The concentration of the sensitizers was similar to that of flavin in parenteral nutrients. The photooxidation of glucose sensitized by flavin mononucleotide or riboflavin was greater than that which was observed in the presence of methylene blue, whereas the isotopic effect of deuterium oxide (D(2)O) was enhanced more substantially in the presence of methylene blue than in the presence of flavins. These results show that methylene blue exerts its action through singlet oxygen and that at a high substrate concentration (as was used in this work) flavin mononucleotide and riboflavin act preferentially as type I sensitizers. In the flavin photosensitized processes, the presence of hydrogen peroxide, superoxide anion, and hydroxyl radical was demonstrated. The photooxidation of glucose is favored by an increase in pH, and it also depends on the energy absorbed by the system. By using a specific reagent for glucose (i.e., o-toluidine), it was possible to quantify the photoconversion of glucose. The results obtained in this work should be considered in the management of glucose-containing parenteral nutrients that are exposed to visible light in the presence of a multivitamin complex containing flavin mononucleotide.

Journal Article↗
PubMed1999

Photochemical and pharmacokinetic properties of selected flavins.

Some photochemical and pharmacokinetic properties of riboflavin, lumiflavin and the 2',3',4',5' tetraacetyl, tetrapropionyl, tetrabutiryl and tetrapalmitoyl esters of riboflavin have been studied. The esters are considerably more photostable than riboflavin but less so than lumiflavin, and appear to be photosensitizers with a behaviour similar to that of riboflavin, promoting photoreactions of biological targets even in the absence of molecular oxygen. The various flavins display important differences in their pharmacokinetic behaviour. Riboflavin, lumiflavin and the short-chain esters (the acetyl and propionyl esters) are rapidly cleared from serum, and recovered in comparable amounts from the liver and kidneys. These results are in agreement with their hydrophilic or moderately hydrophobic character. In contrast, the longer-chain butiryl and palmitoyl esters exhibit a prolonged retention in serum and undergo a significantly larger accumulation in the liver as compared with the kidneys; they are also found in the spleen. In all cases the tissue uptake of these esters becomes appreciable only after 24 h. These results are coherent with the highly hydrophobic character of these esters, which induce a slow release from serum lipoproteins and have a preferential clearance via the bile-gut pathway, showing affinity for the components of the reticuloendothelial system. These long-chain riboflavin esters will probably have a greater and more persistent risk of photoinduced hepatotoxicity than riboflavin, lumiflavin and the short-chain esters.

Animals↗
PubMed1999

VO2 kinetics determined by PRBS techniques differentiate elite endurance runners from elite sprinters.

The aim of the study was to examine whether a measure of oxygen uptake (VO2) kinetics could differentiate between 12 elite male endurance (3000-10,000 m) runners and 12 elite male sprint (100-400 m) runners using a pseudo random binary sequence (PRBS) exercise protocol. All exercise tests were performed on an electrically braked cycle ergometer at a constant pedal frequency of 1 Hz. The PRBS exercise intensities alternated between 25 W and 85 W for three consecutive PRBS cycles of 300 s. VO2 was measured breath-by-breath and results were analysed by Fourier techniques in the frequency domain. Blood lactate concentrations taken pre and post testing were below 2 mM. Significantly greater amplitude components were observed in the endurance runners than sprinters at frequencies 6.7 mHz (6.71 +/- 1.09 and 5.47 +/- 0.95 ml x min(-1) x W(-1), respectively) P<0.05 and 10 mHz (4.97 +/- 0.98 and 3.56 +/- 0.69 ml x min(-1) x W(-1) respectively) P<0.01. Phase shift components were significantly shorter in the endurance runners compared to the sprinters at frequency 3.3 mHz (-35.45 +/- 4.31 and -41.26 +/- 5.82 degrees respectively) P<0.05. The results of this study show that VO2 kinetics are differentially faster in elite endurance runners than in elite sprinters. This supports the development of the PRBS technique as a test of sports performance.

Adult↗
PubMed1999

Methanobacterium thermoautotrophicum RNA polymerase and transcription in vitro.

RNA polymerase (RNAP) purified from Methanobacterium thermoautotrophicum DeltaH has been shown to initiate transcription accurately in vitro from the hmtB archaeal histone promoter with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the M. thermoautotrophicum RNAP and TFB has been demonstrated.

Amino Acid Sequence↗
PubMed1999

Early use of inhaled nedocromil sodium in children following an acute episode of asthma.

BACKGROUND: Current guidelines on the treatment of childhood asthma recommend the introduction of an anti-inflammatory drug in children who have persistent symptoms and require regular treatment with a bronchodilator. The efficacy and safety of inhaled nedocromil sodium (Tilade Mint aerosol) administered using a Fisonair spacer at a dose of 4 mg three times daily was compared with placebo in the treatment of asthmatic children aged 6-12 years who are symptomatic and recovering from an acute exacerbation of asthma. METHODS: A group comparative, double blind, placebo controlled trial was performed in children who were recovering from an acute episode of asthma following treatment in the emergency department of the hospital or in children referred from their general practitioner following a wheezing episode and documented evidence of at least two previous episodes of wheezing. A two week baseline period on existing bronchodilator treatment was followed by a 12 week treatment period on either nedocromil sodium (2 mg/puff) or placebo. Both treatments were administered using a Fisonair spacer at a dose of two puffs three times daily. Changes from baseline values in daytime asthma and night time asthma symptom scores, usage of rescue bronchodilators, mean peak expiratory flow (PEF) recorded twice daily on diary cards, patients' opinion of treatment, and withdrawals due to treatment failure were measured during the primary treatment period (last six weeks of treatment). RESULTS: One hundred and forty two children aged 6-12 years entered the baseline period. Sixty three were withdrawn due to failure to meet the entry criteria (18) or the criteria for asthma symptom severity (15) or reversibility (9), because they developed uncontrolled asthma (2), because they took disallowed treatment (2), or for other non-trial related reasons (17). Seventy nine patients (46 boys) of mean age 8. 8 years entered the treatment period. There were significant differences in the changes from baseline values during the last six weeks of treatment in favour of nedocromil sodium compared with placebo in the primary variables of daytime asthma and night time asthma, morning and evening PEF, and the usage of rescue inhaled bronchodilators; 53% of patients reported nedocromil sodium to be very or moderately effective compared with 44% placebo. Improvement in asthma symptoms, PEF, and reduction in use of rescue bronchodilators did not reach statistical significance until after six weeks of treatment. Twenty two patients were withdrawn or dropped out during the treatment phase, 12 due to uncontrolled asthma or persistence of asthma symptoms, four due to suspected adverse drug reactions (nedocromil sodium 3 (headaches 2, angio-oedema/urticaria 1), placebo 1(persistent cough)), and six due to non-treatment related reasons. Seventy one adverse events were reported by 27 patients in the nedocromil group and 75 by 30 patients in the placebo group. CONCLUSIONS: Asthma symptoms, use of bronchodilators, and lung function can be improved significantly in children recovering from an acute exacerbation of asthma or wheeze and currently receiving treatment with bronchodilators alone by the addition of inhaled nedocromil sodium at a dose of 4 mg three times daily administered using a Fisonair holding chamber.

Acute Disease↗
PubMed1999

Apoptosis induction in nonirradiated human HL-60 and murine NSO/2 tumor cells by photoproducts of indole-3-acetic acid and riboflavin.

The effect of the photoproducts of indole-3-acetic acid sensitized by riboflavin on nonirradiated human HL-60 and murine NSO/2 tumor cells was studied. Severe damage with a dose-response effect was observed on both cell types. The effect was greater than that previously described for the tryptophan riboflavin photoproducts. Electron microscopy studies and flow cytometry analysis of DNA fragmentation allowed us to conclude that the photoproducts studied in this work induce cell death by an apoptotic mechanism.

Animals↗
PubMed1998

The 2.2 A structure of a permanganate-sensitive DNA site bound by the Epstein-Barr virus origin binding protein, EBNA1.

Epstein-Barr nuclear antigen 1 (EBNA1) binds to four recognition sites in the minimal origin of latent DNA replication of Epstein-Barr virus and activates latent-phase replication of the viral genomes. Two of these EBNA1 binding sites become sensitive to permanganate oxidation when bound by the DNA binding and dimerization domains of EBNA1. We have previously solved the co-crystal structure of this EBNA1 fragment bound to a consensus recognition site that is not sensitive to permanganate oxidation (CS). To understand the structural difference that underlies the permanganate sensitivity of EBNA1 binding sites, we have now solved the crystal structure of the EBNA1 DNA-binding and dimerization domains bound to a permanganate-sensitive site (CSA/T). Comparisons of permanganate-sensitive and insensitive EBNA1-DNA complexes have revealed only minor differences in protein and DNA structures. In the EBNA1-CSA/T structure, interstrand H-bonds for three consecutive base-pairs centered over the permanganate-sensitive thymine base are lengthened relative to the corresponding bonds in the EBNA1-CS complex, and three potential intrastrand H-bonds were observed between adjacent bases. We also observed that both the CS and CSA/T sequences are overwound by EBNA1 in the vicinity of the permanganate-sensitive thymine base. Finally, we show that the permanganate-sensitive thymine base in the CSA/T-EBNA1 complex is more accessible to solvent than the corresponding T in the EBNA-CS complex.

Binding Sites↗