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Biomedical subjects

R Edwards

Publications and source records attributed to R Edwards.

At least 19 recordsLinked to original sources
PubMed1998

Thy-1 is a component common to multiple populations of synaptic vesicles.

Thy-1, a glycosylphosphatidylinositol-linked integral membrane protein of the immunoglobulin superfamily, is a component of both large dense-core and small clear vesicles in PC12 cells. A majority of this protein, formerly recognized only on the plasma membrane of neurons, is localized to regulated secretory vesicles. Thy-1 is also present in synaptic vesicles in rat central nervous system. Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel. These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

Animals↗
PubMed1998

Expression of xenobiotic-metabolizing cytochrome P450s in human pulmonary tissues.

The purpose of the study was to obtain a comprehensive picture of the expression of cytochrome P450s (CYP) in the human lung, broncho-alveolar macrophages (BAM), and peripheral blood lymphocytes. The methods used were reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers and immunohistochemistry with specific anti-peptide antibodies. In RT-PCR, CYPs 1A1, 2B6/7, 2E1, 2F1, 3A5 and 4B1 were detected in cDNA prepared from whole lung tissue. BAMs expressed CYPs 1B1, 2B6/7, 2C, 2E1, 2F1, 3A5 and 4B1. These tissues lacked CYPs 1A2, 2A6, 2D6, and 3A7. In peripheral blood lymphocytes, only CYP1B1 and CYP2E1 mRNAs were consistently detected. In immunohistochemistry with anti-CYP3A antibodies, epithelial staining of CYP3A5 was observed in 100% of individuals, while only about 20% exhibited CYP3A4 staining. CYP3A5 protein was localized in the bronchial wall, bronchial glands, bronchiolar epithelium, alveolar epithelium, vascular endothelium and alveolar macrophages. The results indicate that several different xenobiotic-metabolizing CYPs are present in the human lung, possibly contributing to in situ activation of pulmonary procarcinogens.

Cytochrome P-450 Enzyme System↗
PubMed1998

Purification, regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs.

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.

Amino Acid Sequence↗
PubMed1998

Interaction between iron metabolism and 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice with variants of the Ahr gene: a hepatic oxidative mechanism.

The binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the aryl hydrocarbon (AH) receptor and subsequent changes in gene expression have been studied intensively, but the mechanisms by which these lead to toxicity are unclear. We investigated the influence of iron, previously implicated in TCDD-induced hepatic porphyria, in mice with alleles of Ahr that encode receptors with varied affinity for TCDD. The administration of iron to Ahrb-1 C57BL/6J (AH-responsive) mice before a single dose of TCDD (75 micrograms/kg) markedly potentiated not only the hepatic porphyria but also general hepatocellular damage and elevation of plasma hepatic enzymes. The formation of hydroxylated and peroxylated derivatives of uroporphyrins formed from uroporphyrinogen and the induction of a mu-glutathione transferase (GST) were consistent with the operation of an oxidative mechanism. In a comparison of C57BL/6J mice with Ahrb-2 BALB/c (AH-responsive) and Ahrd SWR and DBA/2 (AH-nonresponsive) mice, iron overcame the weak hepatic porphyria and toxicity responses in BALB/c and SWR strains but not in DBA/2. CYP1A isoforms are strongly implicated in the mechanism of porphyria, but activities were lowered by 20-30% with iron treatment, and a comparison of levels between strains did not fully account for the resistance of DBA/2 mice. Studies with the use of gel shift assays and cytosolic aconitase of the capacity of the iron regulatory protein controlling the translation of some iron metabolism proteins showed a significant difference between C57BL/6J and DBA/2 mice after the administration of TCDD. We conclude that iron potentiates both the hepatic porphyria and toxicity of TCDD in susceptible mice in an oxidative process with disturbance of iron regulatory protein capacity. Iron even overcomes the AH-nonresponsive Ahrd allele in the SWR strain but not in DBA/2 mice, which remain resistant.

Alleles↗
PubMed1997

Substrate and thiol specificity of a stress-inducible glutathione transferase from soybean.

An RT-PCR-derived clone encoding a stress-inducible glutathione transferase (GSTGm1) from soybean has been overexpressed in E. coli. The enzyme was active as the dimer GSTGm1-1 and showed GST and glutathione peroxidase activity toward diverse xenobiotics, including analogues of natural stress-metabolites. The selective herbicides, fomesafen and acifluorfen, were conjugated more actively with homoglutathione (hGSH), the major thiol in soybean, than with glutathione (GSH). No thiol preference was shown with the related herbicide, fluorodifen, while GSH was preferred with metolachlor and most non-herbicide substrates. Similar thiol-dependent specificities were observed in GST preparations from plants of varying GSH/hGSH content.

Cloning, Molecular↗
PubMed1997

Adjusting for non-compliance and contamination in randomized clinical trials.

A method of analysis is presented for estimating the magnitude of a treatment effect among compliers in a clinical trial which is asymptotically unbiased and respects the randomization. The approach is valid even when compliers have a different baseline risk than non-compliers. Adjustments for contamination (use of the treatment by individuals in the control arm) are also developed. When the baseline failure rates in non-compliers and contaminators are the same as those who accept their allocated treatment, the method produces larger treatment effects than an 'intent-to-treat' analysis, but the confidence limits are also wider, and (even without this assumption) asymptotically the efficiencies are the same. In addition to providing a better estimate of the true effect of a treatment in compliers, the method also provides a more realistic confidence interval, which can be especially important for trials aimed at showing the equivalence of two treatments. In this case the intent-to-treat analysis can give unrealistically narrow confidence intervals if substantial numbers of patients elect to have the treatment they were not randomized to receive.

Adult↗
PubMed1997

A prototype simulator for endovascular repair of abdominal aortic aneurysms.

A prototype simulator for training in endovascular repair of abdominal aortic aneurysms (AAA) has been developed. Employing transparent models of human AAA complete with renal, iliac and femoral arteries, this system allows accurate simulation of aortography, road-mapping, catheter guidewire manipulation and stent-graft deployment while obviating the need for ionising radiation.

Aortic Aneurysm, Abdominal↗
PubMed1997

Leptin levels do not change acutely with food administration in normal or obese subjects, but are negatively correlated with pituitary-adrenal activity.

BACKGROUND: Leptin is a peptide secreted by white adipose tissue which has been shown to have a major influence on body weight regulation, while animal studies have revealed widespread interconnections between leptin and other endocrine systems, especially with insulin. However, its acute regulation has been little studied in the human. We have therefore investigated the effect of a 1000 kcal meal and fasting on the levels of leptin, insulin and cortisol, in both normal and obese subjects. SUBJECTS AND DESIGN: We have studied the effect of food and fasting on circulating leptin levels in 20 subjects of normal body mass index (BMI range 18-25) and in a group of 12 moderately-severely obese subjects (BMI range 34-61). We also studied the effect of food and fasting in a patient both before and after the successful removal of a pancreatic insulinoma as a model of excess insulin secretion. RESULTS: Mean leptin levels were significantly higher in the obese than in the lean group (42.7 +/- 3.41 vs 5.35 +/- 1.55 micrograms/l, mean +/- SEM; P < 0.001), and showed a positive correlation with body mass index (r = +0.71; P < 0.001). Frequent (every 20 minutes) sampling for 3 hours after food did not show any acute changes in circulating leptin levels. On the fasting day we observed a small but significant fall in circulating leptin levels in the last 4 hours of a 20-hour fast in our subjects as a group (92 +/- 0.03% of basal, P = 0.03); however, in the lean subjects the fall was greater (86 +/- 0.04% of basal, P = 0.02) than in the obese, where it did not reach statistical significance (96 +/- 0.05% of basal). Pre-meal and peak insulin levels showed a positive correlation with circulating mean leptin levels (r = +0.65; P < 0.001 and r = +0.78; P < 0.001, respectively) in all subjects, while pre-meal and peak serum cortisol levels showed an inverse relation with leptin levels (r = -0.53; P = 0.002 and r = -0.41; P = 0.02, respectively); this effect was independent of BMI in the obese subjects. In the patient with the insulinoma the markedly elevated insulin and leptin levels measured before the operation returned to normal after removal of the tumour, in accord with reports of experimental animal data that long-term insulin excess per se is associated with increased circulating leptin concentrations. CONCLUSION: Leptin is a robust indicator of BMI and insulin levels, both basal and stimulated, but does not change acutely following food. Fasting causes a proportionately greater decline in leptin levels in lean subjects than in obese subjects. Circulating leptin is inversely correlated with the activity of the hypothalamo-pituitary-adrenal axis: whether this is a direct influence of leptin on hypothalamo-pituitary-adrenal activity, or whether both are indirect indicators of body fat stores, requires further investigation.

Adult↗
PubMed1997

Functional and genetic studies of the angiotensin II type 1 receptor in pre-eclamptic and normotensive pregnant women.

OBJECTIVE: To examine and compare angiotensin II type 1 receptor genotype and its relationship to platelet angiotensin II binding for pre-eclamptic and normotensive pregnant women. DESIGN: In a case-control study, 43 pre-eclamptic women and 83 normotensive women were genotyped at the angiotensin II type 1 receptor gene locus. Platelet angiotensin II binding was measured for a subset of 11 pre-eclamptic and 57 normotensive pregnant women. We genotyped 162 healthy blood donors also, to examine the allelic background and patterns of linkage disequilibrium in the Nottingham population. METHODS: Patients were recruited during pregnancy using a rigorous definition of pre-eclampsia. DNA was extracted from peripheral venous blood and genotyped at six previously described diallelic polymorphisms in the angiotensin II type 1 receptor gene, using competitive allele-specific oligonucleotide hybridization, and at a dinucleotide repeat polymorphism in the 3' flanking region of the gene. Platelet angiotensin II binding and plasma angiotensin II concentrations were determined for peripheral venous blood. RESULTS: Normotensive pregnant women homozygous for cytosine at nucleotide 573 had significantly higher levels of platelet angiotensin II binding than did heterozygous women and women homozygous for thymidine at this site. Pre-eclamptic women had significantly higher levels of platelet angiotensin II binding than did normotensive pregnant women. The frequencies of allelic variants did not differ significantly between normotensive and pre-eclamptic women. CONCLUSION: The physiological regulation of platelet angiotensin II type 1 receptor expression in normal pregnancy is determined in part by angiotensin II type 1 receptor genotype. There was no evidence that the polymorphisms in the angiotensin II type 1 receptor gene were associated with pre-eclampsia.

Adult↗
PubMed1997

Resistance to beta-lactam antibiotics in Bacteroides spp.

Bacteroides spp., particularly B. fragilis, are well-recognised bacterial pathogens. Production of the typical beta-lactamases of Bacteroides restricts the therapeutic use of beta-lactam agents mainly to the beta-lactamase inhibitor combinations and carbapenems. These compounds have the advantage of broad-spectrum activity and the ability to combat polymicrobial infections. Resistance of Bacteroides spp. to beta-lactam antibiotics appears to be increasing, largely because of an overall increase in beta-lactamase activity. There has been a rise in the prevalence of isolates showing high-level production of typical Bacteroides beta-lactamases and an increase in reports other potent beta-lactamase types. In the case of B. fragilis, metallo-enzymes are a particular threat to current therapeutic practice, as they are not inhibited by common beta-lactamase inhibitors and are able to hydrolyse carbapenems. The presence of permeability barriers may confer low-level beta-lactam resistance and supplement the effect of beta-lactamase activity. There are also sporadic reports of loss of beta-lactam activity because of reduced affinity of the penicillin-binding proteins.

Anti-Bacterial Agents↗
PubMed1997

Genetic differentiation of Australian isolates of Mycobacterium tuberculosis by pulsed-field gel electrophoresis.

As part of an epidemiological study of tuberculosis in Australia, 84 isolates of Mycobacterium tuberculosis from patients were analysed by pulsed-field gel electrophoresis (PFGE). The isolates were genetically heterogeneous, with 66 different DNA banding patterns obtained following digestion of genomic DNA with Dra1 and 53 patterns with Xba1. When the results were compared with those previously obtained in restriction fragment length polymorphism analysis (RFLP), in 87% of cases the results with Dra1 were consistent with those obtained with insertion sequence IS6110 as a probe in RFLP. However, PFGE was able to differentiate four of eight isolates which were identical with IS6110 typing. The high polymorphism amongst strains and the high average age of the patients (51 years) suggested that most organisms were cultured from patients who had reactivation of existing infections. Isolates with identical DNA patterns were found in different states of Australia, but no one strain predominated in any area. This suggests that tuberculosis has been introduced into Australia from various sources.

Adolescent↗
PubMed1997

The effect of zinc ions on the activity of metallo-beta-lactamases of Bacteroides fragilis.

The zinc supplement required to achieve the maximum activity of metallo-beta-lactamases from 12 Bacteroides fragilis isolates was investigated. Changes in absorbance of imipenem in a spectrophotometric assay with crude cell extracts were performed in the presence and absence of various concentrations of zinc sulphate. The greatest degree of imipenem hydrolysis was seen with the addition of between 50 and 500 microM zinc sulphate, and the degree of stimulation of enzyme activity in the strains tested varied six-fold. Increasing the zinc sulphate concentration resulted in inhibition of hydrolysis with extracts of low enzymic activity. These findings indicate the importance of determining the optimal zinc concentration for each strain tested in kinetic studies of metallo-beta-lactamases.

Bacteroides fragilis↗
PubMed1997

Development of a PCR assay for rapid diagnosis of Mycobacterium ulcerans infection.

The diagnosis of Mycobacterium ulcerans infection is hampered by the slow growth of the bacterium in culture, resulting in a delay of several months before a specific diagnosis can be obtained. In addition, M. ulcerans cannot be isolated from water even when there is convincing epidemiological evidence implicating this as the source of infection. The aim of the present study was to develop a PCR assay to circumvent the problems of delayed diagnosis and insensitivity of standard bacterial culture for M. ulcerans. For the PCR, we isolated an M. ulcerans-specific DNA fragment, 1,109 bp long, which is repeated at least 50 times throughout the genome. Use of this sequence as a target for PCR allowed us to detect as few as 2 molecules of genomic DNA in vitro. The PCR was used to detect M. ulcerans DNA in fresh tissue and paraffin-embedded sections from all seven patients with culture-confirmed cases of infection.

Base Sequence↗