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P Macpherson

Publications and source records attributed to P Macpherson.

At least 19 recordsLinked to original sources
PubMed2000

Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents.

We investigated the relationship between DNA cytosine methylation and the expression of two genes associated with resistance to DNA methylation damage. Variants of RajiMex- cells acquired resistance to N-methyl-N-nitrosourea by either reactivating a previously silent O6-methylguanine-DNA methyltransferase (MGMT) gene or by repressing the hMSH6 mismatch repair gene. DNA sequencing and measurements of mRNA and enzyme levels revealed that MGMT activity was not correlated with methylation of the core MGMT promoter. Treatment with the demethylating agent 5-azadeoxycytidine reduced MGMT mRNA and enzyme levels, indicating that methylation of some nonpromoter sequences may be required for MGMT gene expression. In contrast, both hMSH6 mRNA and protein levels were increased by 5-azadeoxycytidine treatment of an N-methyl-N-nitrosourea-resistant variant that did not express detectable hMSH6, which implies that this gene was transcriptionally silenced by cytosine methylation. This could be substantiated by in vitro modification of the CpG sites in the hMSH6 promoter with restriction methylase M.SssI, which abolished the transcription of a reporter gene under its control in a transient transfection assay. Taken together, our data show that treatment with chemical methylating agents alters gene expression patterns through increased CpG methylation of genomic DNA, and thereby permits the emergence and selection of clones that are resistant to these agents due to increased repair or tolerance of O6-methylguanine.

Alkylating Agents↗
PubMed1999

The cytotoxicity of DNA carboxymethylation and methylation by the model carboxymethylating agent azaserine in human cells.

Carboxymethylating agents are potential sources of endogenous DNA damage that have been proposed as possible contributors to gastrointestinal carcinogenesis. The cytotoxicity of the model DNA carboxymethylating agent azaserine was investigated in human cells. Expression of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) did not affect sensitivity to the drug in two related Raji Burkitt's lymphoma cell lines. DNA mismatch repair-defective variants of Raji cells which display increased tolerance to DNA methylation damage were not selectively resistant to azaserine. Complementary results were obtained with a second carboxymethylating agent, potassium diazoacetate. In contrast, lymphoblastoid cell lines representative of each of the xeroderma pigmentosum complementation groups, including the variant, were all significantly more sensitive to azaserine than nucleotide excision repair-proficient cells. The hypersensitivity of XP cells was not due to systematic differences in the concentrations of intracellular thiol compounds or related thiol metabolizing enzymes. The data indicate that of the two types of potentially lethal DNA damage which azaserine introduces, carboxymethylated bases and O(6)-methylguanine, the former are repaired by nucleotide excision repair and are a more significant contributor to azaserine lethality in human cells.

Adenine↗
PubMed1998

Frameshift mismatch recognition by the human MutS alpha complex.

DNA mismatch binding by an extensively purified hMutS alpha mismatch recognition complex was investigated using a bandshift assay. The complex bound to G. T mispairs and to looped structures containing an unpaired single or two adjacent bases. A CA loop was preferentially recognised if the unpaired bases formed part of a repeated sequence. In general, single base loops were also more favourably recognised by hMutS alpha when present in monotonic runs of two to five. In one series of substrates, based on a known hotspot for frameshift mutations in the hypoxanthine-guanine phosphoribosyltransferase gene, in which different length G, A, C or T tracts were flanked by closely similar sequences, an A loop was bound preferentially in the absence of adjacent As and a C loop in the absence of adjacent Cs. This preferential binding was influenced by the base immediately 5' to the loop. Thus, while repeated regions generally favour recognition of single base loops by hMutS alpha, other factors related to local sequence may influence this interaction.

Binding Sites↗
PubMed1997

Mismatch repair defects and O6-methylguanine-DNA methyltransferase expression in acquired resistance to methylating agents in human cells.

Fifteen variants with >/=30-fold resistance to N-methyl-N-nitrosourea were isolated from the Burkitt's lymphoma Raji cell line. Eight had received a single treatment with a highly cytotoxic dose. The remainder, including the previously described RajiF12 cell line, arose following multiple exposures to initially moderate but escalating doses. Surprisingly, methylation resistance arose in three clones by reactivation of a previously silent O6-methylguanine-DNA methyltransferase gene. Five clones, including RajiF12, displayed the microsatellite instability and increased spontaneous mutation rates at the hypoxanthine-guanine phosphoribosyltransferase locus, consistent with deficiencies in mismatch repair. Defects in either the hMutSalpha or hMutLalpha mismatch repair complexes were identified in extracts of these resistant clones by in vitro complementation using extracts from colorectal carcinoma cell lines. Defects in hMutLalpha were confirmed by Western blot analysis. Remarkably, five methylation-resistant clones in which mismatch repair defects were demonstrated by biochemical assays did not exhibit significant microsatellite instability.

Blotting, Western↗
PubMed1996

Processing of O6-methylguanine by mismatch correction in human cell extracts.

Human cell extracts perform an aberrant form of DNA synthesis on methylated plasmids [1], which represents processing of O6-methylguanine (O6-meG). Here, we show that extracts of colorectal carcinoma cells with defects in the mismatch repair proteins that normally correct replication errors do not carry out this synthesis. hMSH2-defective LoVo cell extracts (hMSH for human MutS homologue) performed O6-meG-dependent DNA synthesis only after the addition of the purified hMutS alpha mismatch recognition complex. Processing of O6-meG by mismatch correction requires PCNA and therefore probably DNA polymerase delta and/or epsilon. Mismatch repair-defective cells withstand O6-meG in their DNA [2], making them tolerant to methylating agents. Methylation-tolerant HeLaMR clones, with a mutator phenotype and a defect in either mismatch recognition or correction in vitro, also performed little O6-meG-dependent DNA synthesis. Assays of pairwise combinations of tolerant and colorectal carcinoma cell extracts identified hMLH1 as the missing mismatch repair function in a group of tolerant clones. The absence of processing by extracts of methylation-tolerant cells provides the first biochemical evidence that lethality of DNA O6-meG derives from its interaction with mismatch repair.

Cell Extracts↗
PubMed1996

hMSH2-independent DNA mismatch recognition by human proteins.

Two distinct mismatch binding activities are detected using bandshift assays with human cell extracts and DNA with mispairs at defined positions. One requires hMSH2 protein and is absent from extracts of LoVo cells, which contain a partial deletion of the hMSH2 gene. The second activity is independent of hMSH2 and is present at normal levels in LoVo and three other cell lines, which are defective in in vitro hMSH2-dependent binding. The two mismatch recognition activities are distinguished by their sensitivity to polycations and can be resolved by chromatography on MonoQ. hMSH2-independent activity has been purified extensively from wild-type cells and from a cell line deficient in hMSH2-dependent binding. The purified material preferentially recognizes A-C, some pyrimidine-pyrimidine mismatches, and certain slipped mispaired structures. Binding exhibits some sequence preferences. The similar properties of the two mismatch binding activities suggest that they both contribute to mismatch repair.

Adenocarcinoma↗
PubMed1994

The design and construction of a medical simulation model.

This paper describes the design, construction and validation of a probabilistic simulation model of patients who present with abdominal pain. The model incorporates text-book medical knowledge, clinical judgment, and statistics collected from real cases. The knowledge representation combines techniques of Bayesian network modelling with ideas of logistic discrimination. The model is shown to generate convincing, realistic cases; large numbers of artificial cases with no missing observations can be generated quickly. This should make the model a useful tool for investigating factors which limit achievable computer accuracy in the diagnosis of abdominal pain.

Abdominal Pain↗
PubMed1994

Surfactant proteins A (SP-A) and D (SP-D): levels in human amniotic fluid and localization in the fetal membranes.

Surfactant proteins A (SP-A) and D (SP-D) are major proteins, in the lung, which are composed of collagenous and globular domains. They show an overall similarity to the serum complement protein Clq, which is involved in the initiation of antibody-dependent defence mechanisms. Both SP-A and SP-D were detected, immunochemically, in amniotic fluid as early as 26 weeks gestation and, as expected, SP-A levels rose sharply from 32 weeks towards term. By contrast, SP-D levels in the same samples rose only moderately. Immunochemistry of paraffin sections of fetal membranes, revealed the presence of both SP-A and SP-D in the amniotic epithelium and chorio-decidual layers. SP-A and SP-D are both lectins and therefore they may play a role in the antibody-independent recognition and clearance of pathogens in the amniotic fluid.

Amniotic Fluid↗
PubMed1994

Diffuse brain swelling after head injury: more often malignant in adults than children?

A series of 118 patients with diffuse traumatic brain swelling was studied retrospectively in order to compare the clinical findings in children with those in adults, and to determine the occurrence of neurological deterioration and outcome. The computerized tomography (CT) picture of absent third ventricle and basal cisterns was used to identify the cases. Although this condition has been associated with children, we found the same number of children and adults (59 cases each). Secondary deterioration (decline in consciousness, the development of new focal neurological signs, or an increase in intracranial pressure) occurred in 40% of cases and was more common in adults than children. Features that were significantly associated with deterioration were the presence of prolonged coma (> 1 hour) after the injury, CT signs of diffuse axonal injury or subarachnoid hemorrhage, or a recorded episode of hypotension. A moderate or good recovery at 6 months was achieved by 70 patients (59%), but 45 patients had a poor outcome (severe disability in nine, vegetative state in three, and death in 33) and this was often a consequence of secondary deterioration. In three patients, the outcome was not known. The combination of a severe initial injury, secondary insult, and diffuse swelling is associated with a poor outlook, particularly in adults. The CT appearance of diffuse swelling may develop more readily in children because of the lack of cerebrospinal fluid available for displacement. In children, diffuse swelling may have a relatively benign course unless there is a severe primary injury or a secondary hypotensive insult.

Adolescent↗
PubMed1994

Case-based explanation for medical diagnostic programs, with an example from gynaecology.

One of the most accountable methods of providing machine assistance in medical diagnosis is to retrieve and display similar previously diagnosed cases from a database. In practice, however, classifying cases according to the diagnoses of their nearest neighbours is often significantly less accurate than other statistical classifiers. In this paper the transparency of the nearest neighbours method is combined with the accuracy of another statistical method. This is achieved by using the other statistical method to define a measure of similarity between the presentations of two cases. The diagnosis of abdominal pain of suspected gynaecological origin is used as a case study to evaluate this method. Bayes' theorem, with the usual assumption of conditional independence, is used to define a metric on cases. This new metric was found to correspond as well as Hamming distance to the clinical notion of "similarity" between cases, while significantly increasing accuracy to that of the Bayes' method itself.

Abdomen, Acute↗
PubMed1993

O6-methylguanine residues elicit DNA repair synthesis by human cell extracts.

We have investigated the processing of O6-methylguanine (O6-MeGua) in plasmid DNA by extracts of human cells defective in O6-MeGua-DNA methyltransferase. Cell extracts of HeLaMR cells performed viral T antigen-independent DNA synthesis on plasmids that had been treated with low concentrations of methylating agents. The in vitro DNA synthesis was non-semiconservative and depended on the presence of O6-MeGua in the substrate. The involvement of DNA polymerase delta or epsilon and proliferating cell nuclear antigen but not single-strand binding protein was indicated by partial fractionation, inhibitor, and antibody studies. Processing of O6-MeGua is not via the UV nucleotide excision repair pathway since additional component(s) are apparently required to perform repair synthesis on the methylated substrate. This is the first direct demonstration of DNA repair synthesis provoked by O6-MeGua in DNA. Since O6-MeGua is not excised from DNA by Mex- cells, it represents a novel type of processing of the methylated base that may be involved in its cytotoxicity.

Antibodies↗
PubMed1993

A probabilistic rule-based expert system.

This paper explores a medical expert system combining techniques of Bayesian network modelling with ideas of weighted inference rules. The weights of the individual rules can be estimated objectively from a training set of actual cases; and they can be used in a Monte Carlo stimulation to estimate objectively conditional probabilities of diagnosis given particular combinations of symptoms. The paper describes and evaluates a medical expert system built according to this design. The diagnostic accuracy of the program was found to be similar to that obtained through the usual application of Bayes theorem with the assumption of conditional independence of symptoms given disease, even though the Bayesian classifier has more than 70 times as many numerical parameters. The method may be promising in cases where small training sets do not permit accurate estimation of large numbers of parameters.

Abdominal Pain↗
PubMed1992

Indications for and accuracy of magnetic resonance imaging and computed tomography in orbital disease.

All patients referred for orbital imaging to the neuroradiology department of the Institute of Neurological Sciences in Glasgow over a three year period were enrolled in the study and were scheduled to undergo both magnetic resonance imaging and computed tomography. A total of 101 of the 110 referred patients were deemed suitable for analysis. Details of key presenting symptoms, signs, and a pre-imaging diagnosis were recorded prospectively. A final diagnosis was obtained by histology in 65% of cases with an orbital abnormality, by a minimum of one year of clinical review in 19.5%, by response to antibiotic or steroid therapy in 8.5%, or by conclusive investigations such as carotid angiography in in 7% of patients, 29% of the patients had no detectable orbital disease despite a minimum one years' follow-up, and so were regarded as a "normal" group. The images were interpreted prospectively by separate masked observers. The diagnostic accuracies of the two techniques were compared to the final diagnosis. The two imaging methods were shown to be comparable in overall diagnostic accuracy, with a small and statistically non-significant advantage held by magnetic resonance imaging. Interpretation of the two investigations gave more accurate information in different types of disease.

Academies and Institutes↗
PubMed1991

The value of pre-operative embolisation of meningioma estimated subjectively and objectively.

In a personal series a comparison has been made of the subjective and objective assessments of benefit between a group of patients in whom preoperative embolisation for meningioma was performed and a group, referred during the same timescale, in whom embolisation was either not possible or not appropriate. Preoperative difficulty with bleeding was experienced in 25% of the embolisation group and in 62% of the non-embolised group. Blood replacement overall was less in the embolised patients. Surgical complications occurred in 21% of those embolised but in 54% of those in whom embolisation had not been performed. At follow-up the outcome was rated as good in 79% in the embolisation group as opposed to 58% in the non-embolised group. Preoperative embolisation was therefore beneficial.

Adult↗
PubMed1991

Supplementary CT temporal lobe cuts confer no worthwhile benefit.

The value of angled temporal lobe cuts as a supplement to conventional head computed tomography (CT) has been assessed by comparing the diagnostic yield of standard axial and specific temporal lobe images (TLCT) in 62 patients with temporal lobe epilepsy and 87 with Alzheimer-type senile dementia. Fewer than one patient in six had structural abnormality in the temporal lobe most readily demonstrated by axial CT. Five patients with epilepsy and ten with dementia had changes demonstrated only by TLCT, reported on by one or other of a pair of observers. However such changes were of dubious clinical relevance, or arose as a result of artefact. In one patient with epilepsy and underlying neoplasm, axial CT was positive and TLCT false-negative. The routine addition of temporal lobe cuts to a conventional axial examination confers no added benefit to justify the prolonged examination time and increased radiation dose to the lens of the eye.

Alzheimer Disease↗
PubMed1991

Gadolinium DTPA enhanced magnetic resonance imaging in acute head injury.

We studied the effect of contrast enhancement on magnetic resonance imaging (Gadolinium DTPA Magnetic Resonance) in 10 patients with a recent head injury. The use of contrast did not increase the number of traumatic lesions identified and we did not detect evidence of altered blood brain barrier permeability in any of the 7 patients, who had a total of 27 lesions, imaged between one and 4 days after injury. Enhancement was found in each of 3 patients imaged 6 or more days after injury. These findings suggest that traumatic cortical and intraparenchymal lesions are not associated with increased cerebrovascular permeability within the first 96 hours of a head injury.

Adult↗
PubMed1991

MRI in the management of suspected cervical spondylotic myelopathy.

One hundred and two patients with suspected cervical spondylotic myelopathy were prospectively investigated using MRI as the initial imaging technique. The aim was to discover if clinicians could manage patients with MRI alone, or if they would find a second investigation necessary. Eighty two patients were managed using MRI alone, 34 of whom were treated surgically. Twenty patients had a second investigation: a myelogram in 18 and a CT myelogram in two. This was performed in nine patients to exclude structural pathology in the thoracic or lumbar region (which was not examined with MRI), and in 11 to obtain more specific information about the cervical region. Only five of these 20 patients had surgical treatment. The diagnosis changed after the second investigation in four patients, but management was not influenced in any of these. MRI is a satisfactory alternative to myelography for most patients with suspected cervical spondylotic myelopathy.

Adult↗