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Biomedical subjects

P Vaughan

Publications and source records attributed to P Vaughan.

At least 19 recordsLinked to original sources
PubMed2001

Purification and some characteristics of a recombinant dimeric rhizobium meliloti beta-galactosidase expressed in escherichia coli.

A recombinant Rhizobium meliloti beta-galactosidase was purified to homogeneity from an Escherichia coli expression system. The gene for the enzyme was cloned into a pKK223-3 plasmid which was then used to transform E. coli JM109 cells. The enzyme was purified 35-fold with a yield of 34% by a combination of DEAE-cellulose (pH 8.0) and two sequential Mono Q steps (at pH 8.0 and 6.0, respectively). The purified enzyme had an apparent molecular mass of 174 kDa and a subunit molecular weight of 88 kDa, indicating that it is a dimer. It was active with both synthetic substrates p-nitrophenyl beta-D-galactopyranoside (PNPG) and o-nitrophenyl beta-D-galactopyranoside (ONPG) with K(m)(PNPG) and K(m)(ONPG) of 1 mM at 25 degrees C. The k(cat)/K(m) ratios for both substrates were approximately 70 mM(-1) sec(-1), indicating no clear preference for either PNPG or ONPG, unlike E. coli beta-galactosidase. After non-denaturing electrophoresis, active beta-galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) or 6-bromo-2-naphthyl beta-D-galactopyranoside (BNG) and diazo blue B.

Journal Article↗
PubMed2001

Rapid detection of the R408W and I65T mutations in phenylketonuria by glycosylase mediated polymorphism detection.

Mutation detection methods based upon chemical or enzymatic cleavage of DNA offer excellent detection efficiencies coupled with high throughput and low unit cost. We describe the application of the novel technique of Glycosylase Mediated Polymorphism Detection (GMPD) to the detection of two of the most common mutations of the PAH gene in the Irish population that cause phenylketonuria (PKU), R408W and I65T, which occur at relative frequencies of 41.0% and 10.4% respectively. GMPD assays for R408W and I65T were developed permitting fluorescent detection of cleavage products on the ALFexpresstrade mark automated DNA sequencer. The method was validated by screening a panel of PKU patients whose mutant genotypes had previously been characterised by standard methods. It also proved possible to perform multiplex detection of the two mutations by co-electrophoresis of GMPD products. GMPD is a rapid and robust method for the detection of the R408W and I65T mutations, whose key advantage lies in its use of a pair of enzymes with high cleavage efficiency to detect a number of mutations as compared to the use of individual digestions with a range of specific restriction endonuclease enzymes. Hum Mutat 17:432, 2001.

Alleles↗
PubMed2001

A magnetic resonance study of complicated early childhood convulsion.

OBJECTIVES: The relation between complicated early childhood convulsion (ECC) and adult epilepsy is unclear, although a history of complicated ECC is obtainable in half of adults with epilepsy associated with hippocampal sclerosis. It is not known if the ECC is a marker of pre-existing brain damage or is itself harmful to the developing brain. The objective of the study was to assess the extent of structural brain abnormality present soon after a first complicated early childhood convulsion with a view to obtaining data which might contribute to an understanding of whether such abnormalities were likely to be pre-existing or caused by the convulsion. METHODS: Children under the age of 5 years were recruited into the study after their first complicated febrile or non-febrile ECC. None had previously experienced an epileptic seizure. All underwent MRI of the brain within 14 days. Hippocampal volumes and T2 relaxation times were measured. The results were compared with a neurological control group of children without gross structural abnormalities of the neocortex undergoing MRI of the brain for reasons other than epilepsy. RESULTS: Eighteen patients and 10 control subjects were recruited into the study. One patient was subsequently excluded because of EEG and clinical evidence of benign childhood epilepsy. Nine patients had volumetric evidence of significant hippocampal volume asymmetry (3 SD from the mean of the control group), although in only three of these was the asymmetry apparent on visual inspection of the MRI. Three patients had extrahippocampal neuropathology. None of the control subjects had significant hippocampal volume asymmetry (p<0.001). T2 relaxometry showed no evidence that postictal hippocampal oedema contributed to the asymmetry. CONCLUSIONS: There is a high prevalence of structural brain abnormalities in children within 2 weeks of the first complicated early childhood convulsion, including significant hippocampal asymmetry unrelated to oedema. This does not exclude a damaging effect of complicated ECC on the brain, but suggests that in at least some patients the complicated ECC is the result of pre-existing brain abnormalities.

Brain↗
PubMed2000

Gamma knife radiosurgery as a primary treatment for prolactinomas.

OBJECT: The purpose of this study was to estimate the efficacy of gamma knife radiosurgery (GKS) in controlling tumor growth and endocrinopathy associated with prolactinomas. METHODS: Between 1993 and 1997, 164 of 469 patients with pituitary adenomas treated by GKS harbored prolactinomas. The dose to the tumor margin ranged from 9 to 35 Gy (mean 31.2 Gy), and the visual pathways were exposed to a dose of less than 10 Gy. The mean tumor diameter was 13.4 mm. The mean follow-up time for 128 cases was 33.2 months (range 6-72 months). Tumor control was observed in all but two patients who underwent surgery 18 and 36 months, respectively, after GKS. Clinical cure was achieved in 67 cases. Clinical improvement was noted with a decrease in the hyperprolactinemia after GKS. Nonetheless, in 31 (29%) of 108 patients who were followed for more than 2 years no improvement in serum prolactin levels was demonstrated, although this could be normalized by bromocriptine administration after treatment. Nine infertile women became pregnant 2 to 13 months after GKS and all gave birth to normal children. There was no visual deterioration related to GKS. Five women experienced premature menopause. In these patients there was subtotal disappearance of the tumor and an empty sella developed. CONCLUSIONS: Gamma knife radiosurgery as a primary treatment for prolactinomas can be safe and effective both for controlling tumor growth and for normalization of prolactin hypersecretion. A higher margin dose (> or = 30 Gy) seemed to be associated with a better clinical outcome. Gamma knife radiosurgery may make prolactinomas more sensitive to the bromocriptine.

Humans↗
PubMed2000

Political economy of tobacco control in low-income and middle-income countries: lessons from Thailand and Zimbabwe. Global Analysis Project Team.

Crucial to the success of the proposed Framework Convention on Tobacco Control will be an understanding of the political and economic context for tobacco control policies, particularly in low-income and middle-income countries. Policy studies in Thailand and Zimbabwe employed the analytical perspective of political economy and a research strategy that used political mapping, a technique for characterizing and evaluating the political environment surrounding a policy issue, and stakeholder analysis, which seeks to identify key actors and to determine their capacity to shape policy outcomes. These policy studies clearly revealed how tobacco control in low-income and middle-income countries is also being shaped by developments in the global and regional political economy. Hence efforts to strengthen national control policies need to be set within the context of globalization and the international context. Besides the transnational tobacco companies, international tobacco groups and foreign governments, international agencies and nongovernmental organizations are also playing influential roles. It cannot be assumed, therefore, that the tobacco control strategies being implemented in industrialized countries will be just as effective and appropriate when implemented in developing countries. There is an urgent need to expand the number of such tobacco policy studies, particularly in low-income and middle-income countries. Comprehensive guidelines for tobacco policy analysis and research are required to support this process, as is a broader international strategy to coordinate further tobacco policy research studies at country, regional and global levels.

Developing Countries↗
PubMed1999

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease.

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Amino Acid Sequence↗
PubMed1999

Glycosylase mediated polymorphism detection (GMPD)--a novel process for genetic analysis.

A process for mutation and polymorphism detection is described here that offers significant advances over current mutation detection systems and that has the potential to significantly enhance molecular genetic analysis of human disease. This novel process is referred to as glycosylase mediated polymorphism detection (GMPD) and exploits the use of highly specific DNA glycosylase enzymes to excise substrate bases incorporated into amplified DNA. Action of the glycosylase leaves the DNA with one or more specific abasic sites which can be cleaved by enzymatic or chemical means. The GMPD process permits detection of polymorphisms and mutations using fragment size analysis or solid phase formats. GMPD is particularly suitable for genotyping of single nucleotide polymorphism (SNP) based markers and also permits efficient scanning of genes for unknown polymorphisms and mutations.

DNA Mutational Analysis↗
PubMed1999

Planning to meet the needs of offenders with mental disorders in the United Kingdom.

During the last decade the planning of services for offenders with mental disorders in the United Kingdom has been geared toward diverting them from the criminal justice system to appropriate levels of psychiatric and social care. Although a seamless service system is yet to be developed, the central government has made a concerted effort to promote a better understanding of the needs of offenders with mental disorders and encourage collaboration between the relevant agencies. A major program of research has been initiated, and local health authorities have been encouraged to use a consortium approach to planning and delivery of specialist services. The authors discuss the activities of the Wessex consortium, composed of five local health authorities and a social services department serving a catchment area with a population of 2.5 million in southern England. The consortium has commissioned needs assessments for all offenders with mental illness from the catchment area and a survey of the resources for secure residential treatment in the region. Based on data from this research, the consortium is planning the development of two long-stay secure units to accommodate offenders with a history of repeated inpatient and prison stays and poor response to previous treatment and rehabilitation efforts.

Commitment of Persons with Psychiatric Disorders↗
PubMed1998

mtDNA replicative potential remains constant during ageing: polymerase gamma activity does not correlate with age related cytochrome oxidase activity decline in platelets.

Progressive age-related oxidative phosphorylation (OxPhos) decline is well known in human tissues. Depletion of mitochondrial DNA (mtDNA) causes OxPhos defects in patients with myopathic syndromes and deficient mtDNA replication has been observed in cells cultured from patients with mitochondrial disease. Patients undergoing treatment for AIDS develop OxPhos defects via mtDNA depletion resulting from inhibition of mtDNA polymerase gamma (Polgamma) by 2'-deoxy 3'-azido thymidine. These findings by others give rise to a possible link between mtDNA replication and bioenergetic decline in disease and during ageing. We have designed an in vitro assay for Polgamma function in small tissue samples to explore this possible link. Platelet homogenate Polgamma showed an activity with a K m of 150 microM (dTTP), a V max of 11.8 pmol/min/mg, inhibited (41% inhibition; 50 microM) by ethidium bromide. Determination of several storage characteristics showed that platelets were a convenient source of Polgamma for assay. Polgamma activity in 45 subjects did not coincide with significant age-related decline (P<0.002; P) observed in cytochrome oxidase (CytOx) activity or with citrate synthase activity. Of the activities studied, the only significant age-wise variation was a 24% CytOx deficiency in elderly (>50; n = 19) compared to young (<51; n = 24) individuals (P<0.01; t). These results suggest a maintenance of total cellular mtDNA Polgamma processive levels during ageing, largely independent of total cellular bioenergetic status or mitochondrial number/density. The processive component of Polgamma is therefore unlikely to make a major contribution to age-related bioenergetic activity decline. This does not, however, preclude the possibility that transient periods of inhibition at crucial points of the cell cycle or development may augment existing intracellular deficiencies. The assay described here greatly facilitates study of Polgamma activity in patients with conditions involving mtDNA depletion or rearrangement.

Adult↗
PubMed1998

A novel process for mutation detection using uracil DNA-glycosylase.

A novel process is presented for the detection of known mutations and polymorphisms in DNA. This process, termed glycosylase mediated polymorphism detection (GMPD) involves amplification of the target DNA using three normal dNTPs and a fourth modified dNTP, whose base is a substrate for a specific DNA-glycosylase once incorporated into the DNA. The work described here utilises uracil DNA-glycosylase as the specific glycosylase and dUTP as the modified dNTP. Primers are designed so that during extension, the position of the first uracil incorporated into the extended primers differs depending on whether a mutation is present or absent. Subsequent glycosylase excision of the uracil residues followed by cleavage of the apyrimidinic sites allows detection of the mutation in the amplified fragment as a fragment length polymorphism. Variation in the sizes of the fragment length polymorphisms generated, can be readily achieved through the use of inosine bases in place of adenine bases in the upper and/or lower primers. The GMPD process is also adaptable to solid phase analysis. The use of the process for detection of mutations in the RYR1 and CFTR genes is demonstrated. Overall, the simplicity, specificity, versatility and flexibility of the GMPD process make it an attractive candidate for both small and large scale application in mutation detection and genome analysis.

Base Sequence↗
PubMed1998

Identification of novel mutations in the ryanodine-receptor gene (RYR1) in malignant hyperthermia: genotype-phenotype correlation.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that is triggered in genetically predisposed individuals by common anesthetics and muscle relaxants. The ryanodine receptor (RYR1) is mutated in a number of MH pedigrees, some members of which also have central core disease (CCD), an inherited myopathy closely associated with MH. Mutation screening of 6 kb of the RYR1 gene has identified four adjacent novel mutations, C6487T, G6488A, G6502A, and C6617T, which result in the amino acid alterations Arg2163Cys, Arg2163His, Val2168Met, and Thr2206Met, respectively. Collectively, these mutations account for 11% of MH cases and identify the gene segment 6400-6700 as a mutation hot spot. Correlation analysis of the in vitro contracture-test data available for pedigrees bearing these and other RYR1 mutations showed an exceptionally good correlation between caffeine threshold and tension values, whereas no correlation was observed between halothane threshold and tension values. This finding has important ramifications for assignment of the MH-susceptible phenotype, in genotyping studies, and indicates that assessment of recombinant individuals on the basis of caffeine response is justified, whereas assessment on the basis of halothane response may be problematic. Interestingly, the data suggest a link between the caffeine threshold and tension values and the MH/CCD phenotype.

Female↗
PubMed1998

Differential messenger ribonucleic acid expression in aggressive versus linear periprosthetic osteolysis.

Osteolysis is a radiographic term used to describe bone resorption adjacent to prosthetic implants. This process involves a spectrum of radiographic presentations, from small generalized linear patterns (linear osteolysis) to larger erosive patterns (aggressive osteolysis). The tissue from aggressive osteolytic lesions from five patients were compared with a series of linear osteolytic lesions from five additional patients. Total ribonucleic acid was extracted from these tissue samples, followed by reverse transcription and amplification by the polymerase chain reaction using a series of primers intended to amplify all ribonucleic acid species. The polymerase chain reaction products were separated by gel electrophoresis and compared by side by side analysis (differential display techniques). Transcription initiation factor IIB and cytokine receptor CRFB4 messenger ribonucleic acid were expressed in four of five patients with aggressive osteolytic lesions, as compared with none of five patients with linear osteolytic lesions. Conversely, nonmuscle myosin heavy chain messenger ribonucleic acid was expressed in five of five patients with linear osteolysis, and in none of the five patients with aggressive osteolysis. Thus, there is a difference in cell behavior between linear and aggressive osteolytic lesions that likely accounts for differences in radiographic appearance. This disparity is likely attributable to differences in local conditions (greater amounts of debris, increasing instability of the implant, or increased fluid pressures within the osteolytic lesions), and differences in host response.

Cytokines↗
PubMed1997

In harm's way.

Explore the source record for details and available documents.

Canada↗
PubMed1996

Finding the heart of medicine at the end of the road in Guatemala.

Canadian physician Peter Vaughan recently participated in a medical mission in Guatemala. He recalled that a visit to the war-torn country 20 years earlier had inspired him to go into medicine; his return visit this past March to share his medical skills reconfirmed his decision.

Adult↗