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Biomedical subjects

A D Cameron

Publications and source records attributed to A D Cameron.

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

An evolutionary approach to the design of glutathione-linked enzymes.

Studies of protein structure provide information about principles of protein design that have come into play in natural evolution. This information can be exploited in the redesign of enzymes for novel functions. The glutathione-binding domain of glutathione transferases has similarities with structures in other glutathione-linked proteins, such as glutathione peroxidases and thioredoxin (glutaredoxin), suggesting divergent evolution from a common ancestral protein fold. In contrast, the binding site for glutathione in human glyoxalase I is located at the interface between the two identical subunits of the protein. Comparison with the homologous, but monomeric, yeast glyoxalase I suggests that new domains have originated through gene duplications, and that the oligomeric structure of the mammalian glyoxalase I has arisen by 'domain swapping'. Recombinant DNA techniques are being used for the redesign of glutathione-linked proteins in attempts to create binding proteins with novel functions and catalysts with tailored specificities. Enzymes with desired properties are selected from libraries of variant structures by use of phage display and functional assays.

Binding Sites↗
PubMed1998

Structure of Escherichia coli ribokinase in complex with ribose and dinucleotide determined to 1.8 A resolution: insights into a new family of kinase structures.

BACKGROUND: D-ribose must be phosphorylated at O5' before it can be used in either anabolism or catabolism. This reaction is catalysed by ribokinase and requires the presence of ATP and magnesium. Ribokinase is a member of a family of carbohydrate kinases of previously unknown structure. RESULTS: The crystal structure of ribokinase from Escherichia coli in complex with ribose and dinucleotide was determined at 1.84 A resolution by multiple isomorphous replacement. There is one 33 kDa monomer of ribokinase in the asymmetric unit but the protein forms a dimer around a crystallographic twofold axis. Each subunit consists of a central alpha/beta unit, with a new type of nucleotide-binding fold, and a distinct beta sheet that forms a lid over the ribose-binding site. Contact between subunits involves orthogonal packing of beta sheets, in a novel dimer interaction that we call a beta clasp. CONCLUSIONS: Inspection of the complex indicates that ribokinase utilises both a catalytic base for activation of the ribose in nucleophilic attack and an anion hole that stabilises the transition state during phosphoryl transfer. The structure suggests an ordered reaction mechanism, similar to those proposed for other carbohydrate kinases that probably involves conformational changes. We propose that the beta-clasp structure acts as a lid, closing and opening upon binding and release of ribose. From these observations, an understanding of the structure and catalytic mechanism of related sugar kinases can be obtained.

Adenylyl Imidodiphosphate↗
PubMed1998

All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly.

The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.

Acetyltransferases↗
PubMed1998

Two sisters with prenatal growth failure, disproportionate short stature, and feeding difficulties.

A familial short stature syndrome is described in two sisters. Clinical features include severe pre- and post-natal growth failure, stridor, feeding difficulties in the first 2 years requiring nasogastric feeding and facial dysmorphism reminiscent of Three M syndrome. Intellectual function is normal. Skeletal surveys show short long bones, small square iliac bones, short femoral necks and vertebral bodies which are short in the antero-posterior diameter with narrowing of the interpedicular distance inferiorly.

Bone Diseases, Developmental↗
PubMed1998

Variations in the management of singleton breech presentation throughout Scotland.

Breech presentation occurs in 3-5% of deliveries and can be managed by either a trial of vaginal breech delivery (TOVBD), external cephalic version (ECV) or Caesarean section. A postal questionnaire was completed by 82% of Scottish consultant obstetricians and revealed wide variations in practice. Eighteen percent never offered ECV. Among those who did consensus was lacking on some contraindications. One-quarter sometimes performed ECV before 37 weeks gestations despite the possibility of spontaneous version. Only 70% restricted ECV to one or more designated operators thereby maintaining levels of expertise. Variations were demonstrated in the use of tocolytics, and pre and post procedure investigations. Following failed ECV 28% considered a repeat attempt and 56% a TOVBD. TOVBD was not offered as first line management by one-fifth of respondents. Those who did varied in the pre-procedure investigations performed. Guidelines are required to ensure safe, consistent practice and avoid unnecessary Caesarean sections.

Breech Presentation↗
PubMed1997

Mutagenesis of residue 157 in the active site of human glyoxalase I.

Met-157 in the active site of human glyoxalase I was changed by site-directed mutagenesis into alanine, glutamine or histidine in order to evaluate its possible role in catalysis. The glyoxalase I mutants were expressed in Escherichia coli and purified on an S-hexylglutathione affinity gel. The physicochemical properties of the mutant proteins were similar to those of the wild-type enzyme. The glutamine mutant exhibited the same high specific activity as wild-type glyoxalase I, whereas the alanine and histidine mutants had approx. 20% of wild-type activity. The kcat/Km values of the mutant glyoxalase I determined with the hemithioacetal adduct of glutathione and methylglyoxal were reduced to between 10 and 40% of the wild-type value. This reduction was due to lower kcat values for the alanine and histidine mutants and a twofold increase in the Km value for the glutamine mutant. With the hemithioacetal of glutathione and phenylglyoxal, the kinetic parameters of the mutants were also of the same magnitude as those of wild-type glyoxalase I. Studies with the competitive inhibitors S-hexyl- and S-benzyl-glutathione revealed that the affinity was reduced to 7-11% of the wild-type affinity for the glutamine and alanine mutants and to 30-40% for the histidine mutant, as measured by a comparison of Ki values. The results show that Met-157 has no direct role in catalysis, but is rather involved in forming the substrate-binding site of human glyoxalase I. The high activity of the glutamine mutant suggests that a structurally equivalent glutamine residue in the N-terminal half of Saccharomyces cerevisiae glyoxalase I may be part of a catalytically competent active site.

Amino Acid Sequence↗
PubMed1997

Crystal structure of human glyoxalase I--evidence for gene duplication and 3D domain swapping.

The zinc metalloenzyme glyoxalase I catalyses the glutathione-dependent inactivation of toxic methylglyoxal. The structure of the dimeric human enzyme in complex with S-benzyl-glutathione has been determined by multiple isomorphous replacement (MIR) and refined at 2.2 A resolution. Each monomer consists of two domains. Despite only low sequence homology between them, these domains are structurally equivalent and appear to have arisen by a gene duplication. On the other hand, there is no structural homology to the 'glutathione binding domain' found in other glutathione-linked proteins. 3D domain swapping of the N- and C-terminal domains has resulted in the active site being situated in the dimer interface, with the inhibitor and essential zinc ion interacting with side chains from both subunits. Two structurally equivalent residues from each domain contribute to a square pyramidal coordination of the zinc ion, rarely seen in zinc enzymes. Comparison of glyoxalase I with other known structures shows the enzyme to belong to a new structural family which includes the Fe2+-dependent dihydroxybiphenyl dioxygenase and the bleomycin resistance protein. This structural family appears to allow members to form with or without domain swapping.

Amino Acid Sequence↗
PubMed1997

Purification, characterization, and crystallization of Escherichia coli ribokinase.

Ribokinase phosphorylates ribose to form ribose-5-phosphate in the presence of ATP and magnesium. The phosphorylated sugar can enter the pentose phosphate pathway or be used for the synthesis of nucleotides, histidine, and tryptophan. Ribokinase belongs to the PfkB family of carbohydrate kinases, for which no three-dimensional structure is currently known. We describe an improved purification protocol for Escherichia coli ribokinase and give evidence from light-scattering and gel filtration studies that the protein forms a dimer in solution. Several types of crystals are also described that have been obtained of apo ribokinase, ribokinase in the presence of ATP, and in a ternary complex with an ATP-analogue and ribose. The latter crystals give the best X-ray diffraction. A complete data set has been collected at the synchrotron source in Hamburg, to 2.6 A resolution using a frozen crystal. The crystals belong to space group P6(1)22 or P6(5)22 with cell parameters a = b = 95 A and c = 155 A.

Crystallography, X-Ray↗
PubMed1997

Transabdominal cervico-isthmic cerclage in the management of cervical incompetence.

The use of transabdominal cervico-isthmic cerclage is described in 13 patients with a diagnosis of cervical incompetence. The patients were recruited from seven Scottish Maternity Units over a period of 10 years. The 13 patients have had a successful pregnancy in 86.6% of pregnancies with this procedure compared with a success rate of 16% in their previous pregnancies. In carefully selected cases transabdominal cervico-isthmic cerclage is a worthwhile procedure in patients with cervical incompetence when the cervix is so damaged that it would be impossible to insert a vaginal suture or when a vaginal suture has previously failed.

Adult↗
PubMed1997

Nitric oxide donors induce ripening of the human uterine cervix: a randomised controlled trial.

OBJECTIVE: To determine whether nitric oxide donors can induce cervical ripening before surgical termination of pregnancy in the first trimester. DESIGN: Prospective, randomised controlled trial. SETTING: Department of Obstetrics and Gynaecology, Royal Infirmary, Glasgow. PARTICIPANTS: Forty-eight primigravid women undergoing surgical termination of pregnancy before 12 weeks of gestation. METHODS: The women were randomised to receive per vaginam before surgery either the nitric oxide donor isosorbide mononitrate, the nitric oxide donor glyceryl trinitrate, the prostaglandin analogue gemeprost, or no treatment. MAIN OUTCOME MEASURES: The cumulative force required to dilate the cervix to 8 mm was measured objectively and the cervical diameter before surgical dilatation was recorded. RESULTS: Following isosorbide mononitrate or gemeprost, a lower cumulative force was required to dilate the cervix to 8 mm and a higher cervical diameter before dilatation was recorded. Pretreatment with glyceryl trinitrate reduced the cumulative force required to dilate the cervix but had no effect on cervical diameter. CONCLUSIONS: Like the prostaglandin analogue gemeprost, the nitric oxide donors isosorbide mononitrate and glyceryl trinitrate can effect cervical ripening. Nitric oxide donors may provide an alternative to prostaglandins for cervical ripening before surgical procedures in the first trimester.

Abortifacient Agents, Nonsteroidal↗
PubMed1997

The changing pattern of eclampsia over a 60-year period.

OBJECTIVE: To determine changes in the incidence and pattern of eclampsia within the same geographical area over a 60-year period. DESIGN: A retrospective, descriptive study of 1259 consecutive women classified, at the time, as having had an eclamptic convulsion between the years 1931 and 1990. SETTING: A large city centre teaching hospital and the surrounding catchment area. MAIN OUTCOME MEASURES: The changes in the incidence and timing of the convulsion and the outcomes for the mother and baby. RESULTS: Over the study period, the incidence of eclampsia fell by more than 90%, from 74.1/10,000 in the 1930s to 7.2/10,000 in the 1980s. Most of the reduction occurred over the first four decades, with little change in the last 20 years. Overall, 44% of the cases of eclampsia occurred in the antenatal period, 33% intrapartum and 23% postpartum. Since the biggest decreases were seen in the incidence of antenatal and particularly intrapartum eclampsia, there has been a relative increase in the proportion of eclampsia occurring postpartum. Maternal death from eclampsia occurred in 15.1% of cases between 1931 and 1940, 13.4% between 1941 and 1950, but fell dramatically to < or = 3.9% after 1950. There has been no maternal death since 1964. Apart from the first decade, postpartum eclampsia was associated with significantly less risk of death to the mother throughout the study period. Perinatal death rate has fallen steadily from 432.6/1000 cases of eclampsia between 1931 and 1940 over the first three decades, to 168.7/1000 between 1961 and 1970. There has been little change since, although a lower proportion of neonatal deaths occurred as stillbirths. CONCLUSIONS: We found a significant reduction in both the incidence of eclampsia and associated morbidity in this population over the last 60 years. This has occurred in association with the introduction of the National Health Service, widespread antenatal care for all and a general improvement in health and welfare. Any further reduction in the incidence in the UK may be difficult to achieve. Since the incidence of eclampsia is now low, efforts should perhaps be directed at minimising the morbidity associated with severe pre-eclampsia rather than prevention of convulsions.

Adult↗
PubMed1997

Human parvovirus B19 infection associated with hydrops fetalis.

Human parvovirus B19 infection has been associated with fetal anaemia, hydrops and in some cases fetal demise. We report our experience with nonimmune hydrops fetalis due to human parvovirus B19 infection in 17 pregnancies which were identified sonographically over a period of 10 years. The only baby who survived received repeated intrauterine transfusions and was normal neurodevelopmentally at 2 years follow-up. Two other fetuses who also received intrauterine transfusion did not survive. All pregnancies complicated with nonimmune hydrops should be investigated by fetal blood sampling looking for the evidence of parvovirus infection. Intrauterine transfusion should be reserved for hydropic fetuses with a low haematocrit. A greater understanding of the natural history of human parvovirus infection is needed prior to deciding the optimum mode of therapy.

Adult↗
PubMed1997

Factors affecting women's knowledge of antenatal serum screening.

Antenatal screening for fetal anomaly is offered routinely in Glasgow. This study assessed pregnant women's knowledge of the test and implications of results. Questionnaires were completed by 574 women. Knowledge of the nature and uses of the test was superior to earlier studies Two-thirds knew that screening was undertaken for Down's syndrome, and 81% for spina bifida. The majority were aware of the sample used, ages invited and gestation at which it was undertaken. Knowledge of the likely results and implications was poorer. Three-quarters were unaware that 10% of results would suggest an increased risk. Half did not realise that positive results could occur without fetal abnormality, or negative results could be falsely reassuring. Socioeconomic deprivation was associated with poorer knowledge but not lower uptake. Written information was associated with superior knowledge and higher uptake. Leaflets should be provided prior to booking, at which time resultant queries can be answered.

Adolescent↗
PubMed1997

Effects of cGMP and the nitric oxide donors glyceryl trinitrate and sodium nitroprusside on contractions in vitro of isolated myometrial tissue from pregnant women.

The purpose of this study was to determine the relaxant effects in vitro of two nitric oxide donors, glyceryl trinitrate and sodium nitroprusside, which are currently available for use in vivo, on contractions of non-labouring myometrium from pregnant women. Since nitric oxide also mediates relaxation by increasing the concentration of cGMP, sensitivity to 8-bromo-cGMP (a cGMP analogue) was also determined. The effects of the K(+)-channel opener lemakalim and of the Ca(2+)-channel blocker nifedipine were studied for comparison. After the addition of glyceryl trinitrate (0.1-100 mumol l-1), sodium nitroprusside (0.1-100 mumol l-1) or 8-bromo-cGMP (0.001-3 mmol l-1), the spontaneous rhythmic contractility of myometrial strips was inhibited in a concentration-dependent manner: the maximum inhibition produced by the highest tested concentration of each drug was 40 +/- 7%, 53 +/- 8% and 39 +/- 8% of the original degree of contraction, respectively. Myometrial contractions were completely abolished by lemakalim and by nifedipine and verapamil at concentrations of > or = 10(-5) mol l-1. The nitric oxide donors, glyceryl trinitrate and sodium nitroprusside, attenuate myometrial contractions and are therefore useful as tocolytic agents. However, at equimolar concentrations in vitro, the ability of glyceryl trinitrate and sodium nitroprusside to attenuate myometrial contractions is less than that of lemakalin, nifedipine and verapamil. Controlled trials are required to determine the side-effects and clinical efficacy of each of these agents in vivo.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

The effect of epidural bupivacaine on the fetal electrocardiogram.

Fetal electrocardiogram waveform analysis was used to assess the effect of epidural bupivacaine on the fetal myocardial conducting system by evaluating its effect on the PR interval, RR interval, T/QRS ratio and the PR-RR correlation coefficient. There were no significant changes in either the PR interval or the PR-RR correlation coefficient. There was a significant increase in the fetal heart rate and a significant fall in the T/QRS ratio. Epidural bupivacaine does not alter fetal myocardial conduction as measured by the PR interval and it does not induce ischaemic cardiac changes as assessed by the T/QRS ratio.

Adolescent↗
PubMed1995

Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate.

BACKGROUND: Glutathione transferases (GSTs) constitute a family of isoenzymes that catalyze the conjugation of the tripeptide glutathione with a wide variety of hydrophobic compounds bearing an electrophilic functional group. Recently, a number of X-ray structures have been reported which have defined both the glutathione- and the substrate-binding sites in these enzymes. The structure of the glutathione-free enzyme from a mammalian source has not, however, been reported previously. RESULTS: We have solved structures of a human alpha-class GST, isoenzyme A1-1, both in the unliganded form and in complexes with the inhibitor ethacrynic acid and its glutathione conjugate. These structures have been refined to resolutions of 2.5 A, 2.7 A and 2.0 A respectively. Both forms of the inhibitor are clearly present in the associated electron density. CONCLUSIONS: The major differences among the three structures reported here involve the C-terminal alpha-helix, which is a characteristic of the alpha-class enzyme. This helix forms a lid over the active site when the hydrophobic substrate binding site (H-site) is occupied but it is otherwise disordered. Ethacrynic acid appears to bind in a non-productive mode in the absence of the coenzyme glutathione.

Apoenzymes↗
PubMed1994

Allosteric activation in Bacillus stearothermophilus lactate dehydrogenase investigated by an X-ray crystallographic analysis of a mutant designed to prevent tetramerization of the enzyme.

The crystal structure of a mutant Bacillus stearothermophilus lactate dehydrogenase, into which an additional loop has been engineered in order to prevent tetramerization of the enzyme, has been solved and refined at 2.4 A. The minimal repeat unit in the crystal is a dimer and the tetramer cannot be generated by any of the crystallographic symmetry operations in P2(1). The loop protrudes out into the solvent, stabilized by a good hydrogen bonding arrangement, and clearly sterically hinders tetramer formation. This is the first structure of B. stearothermophilus lactate dehydrogenase (bsLDH) in which the allosteric activator fructose, 1,6-bisphosphate (FBP) is not present. To investigate the mechanism of allosteric activation in this enzyme we have compared the structure with a ternary complex of B. stearothermophilus lactate dehydrogenase. Many of our observations confirm those reported from a comparison of FBP-bound ternary bsLDH complex with an FBP free LDH from another bacterial source, Bifidobacterium longum. Our results suggest that quaternary structural alterations may have less influence on the mechanism than previously reported. The differences in the quaternary structural behaviour of these two enzymes is discussed.

Allosteric Regulation↗