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R J Edwards

Publications and source records attributed to R J Edwards.

At least 19 recordsLinked to original sources
PubMed2006

Urinary proteomic profiles distinguish between active and inactive lupus nephritis.

OBJECTIVES: Key aims of the treatment of lupus nephritis (LN) are to induce and maintain remission with minimal side effects. However, assessing ongoing renal inflammatory activity is poorly served by current diagnostic tests apart from renal biopsy, but frequent biopsies cannot be justified. Our long-term aim is to identify novel biomarkers from urinary protein profiles to improve diagnosis and monitoring of activity and response to therapy in LN. METHODS: We used surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify biomarkers able to discriminate between urine samples from patients with inactive (n= 49) and active (n= 26) LN. Discriminant function analysis was used to define the minimum number of proteins whose levels best distinguished between the two patient groups. Serial urines of six biopsied patients were studied prospectively, and multiple regression (MR) scores calculated. RESULTS: Proteins with masses of 3340 and 3980 distinguished active from inactive LN with 92% sensitivity and specificity of 92% each. The prospective study of the biopsied patients demonstrated that MR scores could predict both relapse and remission earlier than traditional clinical markers. CONCLUSIONS: SELDI-TOF MS identified potential biomarker profiles strongly associated with activity in LN. Identification of these proteins will allow us to devise specific assays to routinely monitor disease progression, and alter immunosuppressive drug regimens accordingly. These proteins may also play a critical role in the pathogenesis of glomerulonephritis, and could therefore provide targets for therapeutic intervention.

Adult↗
PubMed2006

Monitoring modulators of platelet aggregation in a microtiter plate assay.

Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.

Amino Acid Sequence↗
PubMed2005

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach.

BACKGROUND: Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event. RESULTS: A combined bioinformatics approach of motif prediction and evolutionary and structural analyses identified tyrosines163 and 1856 of the skeletal muscle heavy chain as the leading candidate for the sites of insulin-mediated tyrosine phosphorylation. CONCLUSION: Our work is suggestive that tyrosine phosphorylation of myosin heavy chain, whether in skeletal muscle or in platelets, is a significant event that may initiate cytoskeletal reorganization of muscle cells and platelets. Our studies provide a good starting point for further functional analysis of MHC phosphor-signalling events within different cells.

Algorithms↗
PubMed2003

Differential expression of cytochrome P450 enzymes in cultured and intact foetal rat ventral mesencephalon.

Genetic susceptibility to toxin action leading to nigral cell degeneration in Parkinson's disease (PD) may be dictated by the activity of P450 enzymes in brain. In adult rat brain only CYP2C13/6, CYP2D2, CYP2D5 are found in the substantia nigra. However, little is known concerning the isoforms present in foetal dopaminergic ventral mesencephalon (VM) tissues commonly used to study toxin action. In this investigation, we have determined the expression of P450 enzymes in foetal (VM) slices and in primary cultures of this region. In foetal VM sections immunoreactivity was observed for CYP2C13/6, CYP2D1, CYP2D3, CYP2D4, CYP2D5 and OR. There was no expression of CYP1A2, CYP2B1/2, CYP2C12, CYP2D2 and CYP2E1. In cultured foetal rat VM, immunoreactivity was observed for all P450 enzymes examined, namely CYP1A2, CYP2B1/2, CYP2C13/6, CYP2C12, CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5, CYP2E1 and OR. There were marked differences in the degree of expression of the isoforms of P450, for example CYP2D1 was only weakly expressed in foetal VM sections but expression was strong in VM cultures. The difference between VM slices and primary cultures suggests that the culturing process can induce some P450 enzymes. CYP2D1, CYP2D3, CYP2D4 were expressed in the foetal VM but were not present in adult rat substantia nigra. Further investigation is now required to determine the functional implications as they may confer an altered level of susceptibility to neurotoxins between foetal and adult dopaminergic cells.

Animals↗
PubMed2003

Chronic headaches in adults with spina bifida and associated hydrocephalus.

INTRODUCTION: Adults with spina bifida and associated hydrocephalus are exposed to multiple risk factors for the development of chronic headache. The management of these patients can be complex and misdiagnosis can precipitate unnecessary shunt revision. This study aims to evaluate the usefulness of intracranial pressure (ICP) monitoring as a diagnostic tool in these cases and to look at the causes of chronic headaches and treatment outcomes for this patient population. METHODS: All patients over the age of 18 years with a diagnosis of spina bifida and shunted hydrocephalus who had undergone inpatient or outpatient neurosurgical review within the last 10 years were identified in our hospital database. Case notes were then retrospectively reviewed to identify all patients who had undergone either inpatient or outpatient evaluation of chronic headaches (defined as a headache of at least one month's duration) occurring in the absence of any other symptoms or signs suggestive of raised intracranial pressure (ICP). The incidence, causes, management and outcome of chronic headache in these patients was determined. RESULTS: 42 patients were identified, mean age 30 years (range 18 - 59). All had undergone lifelong follow-up. All had previously undergone shunt insertion for hydrocephalus. 16 had undergone endoscopic third ventriculostomy (ETV). 11 had undergone choroid plexus coagulation. 55 % (23/42) of patients underwent investigation for 1 or more episodes of chronic headache. Recurrent hydrocephalus due to shunt malfunction or ETV failure was excluded by ICP monitoring using either an intraparenchymal transducer or monitoring via a ventricular access device. All patients underwent repeat imaging, using CT and/or MR imaging. Identified causes of headache included: shunt blockage; shunt overdrainage; ETV failure and symptomatic Arnold-Chiari malformation. A history of choroid plexus coagulation (CPC) as an infant was associated with a decreased risk of chronic headache in later life (p = 0.02). In 8 patients no definite cause for headaches was identified, in 4 of these patients symptoms resolved spontaneously, the remainder required specialist pain management. CONCLUSIONS: The aetiology of chronic headaches in this patient group is multifactorial. In the absence of other clinical symptoms or signs of raised ICP, ICP monitoring is an invaluable adjunct to management. 10 % of hydrocephalic adult spina bifida patients required specialist pain management for control of chronic idiopathic headache.

Adolescent↗
PubMed2002

Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells.

1. Cultured hepatic cells have reduced cytochrome P450 (CYP) activities in comparison with human liver, but the mechanism(s) that underlies this circumstance is not clear. We investigated the causes of this low CYP activity by analysing the activity, protein, mRNA and heterologous nuclear RNA contents of the most important CYPs involved in drug metabolism (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) in cultured human hepatocytes, and in HepG2 and Mz-Hep-1 hepatoma cell lines. 2. After 24 h of culture, hepatocytes retained most of their CYP activities and protein contents, but the mRNA decreased 20-fold. However, the mRNA content of most CYPs in 24-h hepatocytes was still 400-fold higher than in hepatoma cells. When we examined the transcriptional activity of the CYP genes, this decreased during culture time in hepatocytes and it was poor in hepatoma cell lines. 3. We investigated the abundance of key hepatic transcription factors that govern CYP transcription (C/EBP-beta: LAP and LIP, HNF-3alpha, HNF-4alpha, RXR-alpha) and observed that the expression of some factors was altered in the hepatoma cells. 4. In conclusion, the loss of biotransformation activity in cultured hepatic cells is caused by a decrease in CYP transcription, which correlates with an alteration in the expression of key transcription factors.

Blotting, Western↗
PubMed2002

Cytochrome P450 3A conjugation to ubiquitin in a process distinct from classical ubiquitination pathway.

We characterize a novel microsome system that forms high-molecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16alpha-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg(2+), or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.

Animals↗
PubMed2002

Antiarrhythmic and anti-ischaemic effects of angina in patients with and without coronary collaterals.

OBJECTIVE: To determine whether the changes in the manifestations of myocardial ischaemia during sequential angina episodes caused by exercise or coronary artery occlusion are collateral dependent. METHODS: 40 patients awaiting percutaneous transluminal coronary angioplasty for an isolated left anterior descending artery stenosis underwent three sequential treadmill exercise tests, with the second exertion separated from the first by 15 minutes, and from the third by 90 minutes; 28 patients subsequently completed two (> 180 s) sequential intracoronary balloon inflations with measurement of collateral flow index from mean coronary artery wedge, aortic, and coronary sinus pressures. RESULTS: On second compared with first exercise, time to 0.1 mV ST depression (mean (SD): 340 (27) v 266 (25) s) and rate-pressure product at 0.1 mV ST depression (22 068 (725) v 19 586 (584) beats/min/mm Hg) were increased (all p < 0.005), while angina and ventricular ectopic beat frequency were diminished (p < 0.05). This advantage, which had waned by the third effort, was independent of collateral flow index. Similarly, at the end of the second compared with the first coronary occlusion, ventricular tachycardia (21% v 0%, p < 0.05), ST elevation (0.47 (0.07) v 0.33 (0.05) mV, p < 0.005), and angina severity (6.1 (0.7) v 4.6 (0.7) units, p < 0.005) were reduced despite similar collateral flow indices. CONCLUSIONS: In patients with coronary artery disease, ventricular arrhythmias, ST deviation, and angina are reduced during a second exertion or during a second coronary occlusion. This protective effect can occur independently of collateral recruitment. These characteristics, together with the breadth and temporal pattern of protection, are consistent with ischaemic preconditioning.

Aged↗
PubMed2002

Fatal instability following "odontoid sparing" transoral decompression of a periodontoid pseudotumour.

Pseudotumour of the craniovertebral junction is an uncommon cause of high cervical myelopathy in the elderly. The anterior transoral approach is the preferred means of accessing these lesions. An "odontoid sparing" transoral approach, in which there is only minimal bone removal, is thought to preserve stability of the craniovertebral junction, obviating the need for posterior stabilisation. This report is of an 82 year old man who developed fatal atlanto-axial instability following an odontoid sparing transoral resection of a pseudotumour. This complication has not previously been described and its occurrence has important implications for the surgical management of this condition.

Aged↗
PubMed2001

Diazinon is activated by CYP2C19 in human liver.

Phosphorothioate compounds are used throughout the world as agricultural and domestic pesticides. Here, the activation of the phosphorothioate diazinon to diazoxon in human liver is described. In an initial study using three human liver microsomal samples, K(m) for diazoxon formation varied markedly (31, 208, and 660 microM; V(max) 1125, 685, and 1028 pmol/min/mg protein, respectively), suggesting the involvement of more than one P450 enzyme. A wide variation in activity was found using 50 microM diazinon as substrate, (11-648 pmol/min/mg protein, n = 15), whereas, with 500 microM, variation was less (164-978 pmol/min/mg protein). Among eight P450-catalyzed reactions, the putative high-affinity component (50 microM diazinon) correlated with S-mephenytoin 4'-hydroxylase activity (r = 0.686, p < 0.01), suggesting the involvement of CYP2C19. The putative low-affinity component (500 microM diazinon) correlated with both S-mephenytoin 4'-hydroxylase (r = 0.714; p < 0.005) and high-affinity phenacetin O-deethylase activity (r = 0.625; p < 0.05). This activity was partially inhibited by furafylline, troleandomycin, and ketoconazole. These data suggest contributions from CYP2C19, CYP1A2, and CYP3A4. None of the inhibitors affected the high-affinity component. Of seven heterologously expressed human P450 enzymes, CYP2C19 activated diazinon (500 microM) at the fastest rate, followed by CYP3A4, CYP1A2, and CYP2C9. Both hepatic microsomal S-mephenytoin 4'-hydroxylase and high-affinity phenacetin O-deethylase activities were strongly inhibited by diazinon (IC50 < 2.5 microM), while no effect was seen on midazolam 1'-hydroxylase activity. These data indicate that CYP2C19 is the major enzyme involved in diazinon activation in human liver, while other enzymes including CYP1A2 may play a more minor role.

Aryl Hydrocarbon Hydroxylases↗
PubMed2001

Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples.

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 microg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 microg/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual's non-workplace exposure to cadmium, or an individual's CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYP1A2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYP1A2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, W/C, and C/C were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations.

Adolescent↗
PubMed2001

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies.

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m). The antibodies, together with an anti-marmoset CYP2E1 antibody, were used to investigate the expression of 10 P450 enzymes in marmosets treated with P450-inducing chemicals. Treatment with phenobarbitone caused CYP2B, CYP2C-2 and CYP3A21 levels to increase, rifampicin caused increases in CYP2B and CYP2C-1 and a decrease in CYP3A21 levels, whereas dioxin caused CYP1A1 and CYP1A2 levels to increase and CYP2E1 levels to decrease. Clofibric acid did not induce any P450. P450 enzyme activities were assessed using 8 different substrates and increases were found after treatment with phenobarbitone, rifampicin, and dioxin. However, due to species differences in substrate selectivity, it proved difficult to ascribe these changes to individual P450 enzymes. Thus, the use of anti-peptide antibodies provides a more informative way of assessing the levels of specific P450 enzymes than enzyme activity measurements.

Amino Acid Sequence↗
PubMed2001

Inhibition of human CYP1A2 activity in vitro by methylxanthines: potent competitive inhibition by 8-phenyltheophylline.

1. Humans are exposed in vivo to methylxanthines by dietary ingestion, as well as from their use as therapeutic agents. The inhibitory effect of a series of these compounds on high-affinity phenacetin O-deethylase activity in the human liver microsomal fraction, a measure of CYP1A2 activity, has been evaluated. 2. Paracetamol, the product of phenacetin O-deethylase activity, was analysed by gas chromatography/negative-ion mass spectrometry using a novel bistrifluoromethylbenzoyl/ trimethylsilyl derivative, and incubation conditions for assessing high-affinity phenacetin O-deethylase activity were examined and optimized. 3. 1-Methylxanthine, caffeine, theophylline, 8-methylxanthine, pentoxyfylline and 3isobutyl-1-methylxanthine caused moderate inhibition with IC50 = 260, 140, 120, 100, 62 and 36 microM respectively. 4. 8-Phenyltheophylline was a potent competitive inhibitor of high-affinity phenacetin O-deethylase activity with an IC50 = 0.7 microM and Ki = 0.11 microM. 5. The specificity of inhibition by 8-phenyltheophylline was assessed by measuring its effect on debrisoquine 4-hydroxylase (CYP2D6), terfenadine hydroxylase (CYP3A4), chlorzoxazone 6-hydroxylase (CYP2E1) and tolbutamide 4-hydroxylase (CYP2C9) activities in human liver microsomal fraction. No inhibition of any of these activities was observed. 6. The potency and specificity of 8-phenyltheophylline as an inhibitor of human hepatic CYP1A2 indicate that the compound may be useful as a chemical inhibitor of this enzyme for further in vitro studies.

Acetaminophen↗
PubMed2001

Crisis in resources: a neurosurgical prospective.

In the light of the recent recommendations of the Society of British Neurological Surgeons on safe neurosurgery, we prospectively audited the work load, availability and efficiency in the use of neurosurgical resources in our department over a 1-month period. There were a total of 130 admissions--70 emergency and 60 elective cases. Twenty-three patients referred as emergencies were denied admission, directly or indirectly, because of shortage of beds. Other emergency cases had to wait unacceptable long periods, up to 17 days, prior to admission for the same reason. For elective cases, there was a 33% cancellation rate due to lack of beds and competition with the emergency cases for the same resources. Overall bed occupancy rate exceeded 100%. We conclude that the current neurosurgical resources are inadequate and over-stretched leaving no safety margin. Neurosurgery as a core emergency service, therefore, should be given priority in NHS resource allocation.

Bed Occupancy↗