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P R Wakeley

Publications and source records attributed to P R Wakeley.

11 recordsLinked to original sources
PubMed2006

Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from Taylorella asinigenitalis.

A discriminatory real time PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and the related species T. asinigenitalis was developed for the direct examination of genital swabs. The 112bp amplicons produced from the two species were discriminated from each other using TaqMan probes labelled with different fluorophores. The TaqMan PCR was shown to be specific for the 16S ribosomal DNA of the two species of taylorella and did not cross-hybridise with the 16S ribosomal DNA of other bacteria tested. Direct amplification from genital swabs was shown to be equally sensitive to that of culture methods. Prevalence in a sample set from The Netherlands was shown to be equivalent to that demonstrated by culture. A companion real time PCR that amplified a fragment of the 16S rDNA gene of equine commensal bacteria was developed to ensure bacterial DNA was extracted from swab material supplied for testing. The use of a rapid and reliable real time PCR for the organism causing CEM should aid the control of this disease.

Animals↗
PubMed2006

Development of a real-time, differential RT-PCR TaqMan assay for lyssavirus genotypes 1, 5 and 6.

A number of RT-PCR methods have been reported for the detection of rabies and rabies-related viruses. Here, a single, closed tube, non-nested RT-PCR TaqMan assay to distinguish between Classical rabies virus and European bat lyssaviruses 1 and 2 in real time is described. The TaqMan assay is rapid, sensitive, specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. It can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. The efficiency and dynamic range of the PCR has been established using isolated viral RNA and cloned control template. Comparative performance of the TaqMan assay against other diagnostic methodologies for the detection of rabies virus has been determined and the assay validated against a panel of archival samples and virus of unknown genotype from both Germany and the Sudan. Despite sequence heterogeneity between the different genotypes in the N-gene, a universal forward and reverse primer set have been designed allowing simplification of previously described assays. Rapid genotyping of two recent EBLV2 cases in the U.K. in Daubenton's bats and a recently imported human case of rabies has been performed using this test.

Animals↗
PubMed2005

Development of a real-time, TaqMan reverse transcription-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6.

Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus.

Animals↗
PubMed2004

Characterisation of a type 2 bovine viral diarrhoea virus isolated from cattle in the UK.

Two genotypes of bovine viral diarrhoea virus (BVDV) are recognised. Type 2 was first recognised when virulent strains caused significant losses among cattle in North America. Subsequently, BVDV type 2 has been found in many other countries, but recent studies have shown that only type 1 BVDV is circulating in the UK herds (sheep and cattle) with type 1a predominating. During routine genotyping of UK BVDV isolates, a type 2 isolate was identified. Phylogenetic analysis of the 5'-untranslated region of the viral genome showed it to be a BVDV type 2a, most similar to a low virulent US strain of BVDV type 2. Antigenic typing with a panel of monoclonal antibodies verified this classification. This is the first confirmed isolation of BVDV type 2 found circulating in the UK.

5' Untranslated Regions↗
PubMed2000

Proteolysis of SpolVB is a critical determinant in signalling of Pro-sigmaK processing in Bacillus subtilis.

SpoIVB is essential for intercompartmental signalling in the sigma(K)-checkpoint of Bacillus subtilis. SpoIVB is synthesized in the spore chamber and is the signal which activates proteolytic processing of pro-sigma(K) to its mature and active form sigma(K). We show here that SpoIVB is a serine peptidase of the SA clan. Expression of SpoIVB in Escherichia coli has shown that SpoIVB is able to self-cleave into at least three discrete products, and in vitro studies have shown cleavage in trans. Autoproteolysis of SpoIVB is tightly linked to the initiation of the two developmental functions of this protein, signalling of pro-sigma(K) processing and a yet, uncharacterized, second function which is essential for the formation of heat-resistant spores. In B. subtilis, SpoIVB is synthesized as a zymogen and is subject to two levels of proteolysis. First, autoproteolysis generating intermediate products, at least one of which is proposed to be the active form, followed by processing by one or more enzymes to smaller species. This could provide a mechanism for switching off the active SpoIVB intermediate(s) and suggests a similarity to other proteolytic cascades such as those found in blood coagulation.

Bacillus subtilis↗
PubMed1999

Characterization of two Bacillus probiotics.

Bacillus subtilis is currently used as an oral probiotic. We examined two commercial B. subtilis probiotic preparations, Enterogermina and Biosubtyl. Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B. subtilis.

Anti-Bacterial Agents↗
PubMed1998

A maize pectin methylesterase-like gene, ZmC5, specifically expressed in pollen.

Pectin methylesterase (PME) is responsible for the demethylation of pectin prior to pectin's degradation by the combined activities of polygalacturonase and pectate lyase. We have differentially screened a maize pollen cDNA library to detect cDNA clones whose genes are specifically expressed in pollen. One group of clones resulting from this screen showed similarity (between 18% and 41% identity) with plant and fungal PMEs. The full-length clone from this group, ZmC5, identifies a small gene family (at least 2 members) when used as a probe on genomic Southern blots. Northern analysis reveals that the ZmC5 transcript is expressed specifically in late pollen development. This tissue-specific gene expression programme is further confirmed in transgenic tobacco plants harbouring ZmC5 promoter/GUS chimeric gene constructs.

Base Sequence↗
PubMed1994

Comparison of the in vitro translated polypeptides from maize shoot, pollen and germinated pollen mRNAs.

We have compared the two-dimensional gel (2D gel) profiles of in vitro synthesised polypeptides from maize shoot with that of pollen, and the latter with that of germinated pollen. In the former comparison, extensive quantitative and qualitative differences in the polypeptides are apparent. We describe an increase in the abundance of the major cytoskeletal protein, alpha tubulin, in pollen compared to shoot. We also show that the electrophoretically separable alpha tubulin polypeptides in ungerminated pollen are not obviously post-translationally modified. In the comparison between the 2D gel profiles of the in vitro synthesised polypeptides from pollen and germinated pollen a number of notable differences are apparent, the possible origins of which are described.

Blotting, Northern↗
PubMed1991

Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization.

The stromelysins are members of a family of extracellular matrix metalloproteinases. These enzymes may erode the connective tissue in atherosclerotic plaques, leading to fissuring and acute thrombotic events. Cell-specific stromelysin expression in human atherosclerotic plaques was studied by in situ hybridization and immunocytochemistry. Sections were taken from nine coronary arteries: eight with well-established plaques and one normal. Unambiguous signals were seen in five plaques, two were inconclusive, and the remaining sample was negative, as was the normal coronary artery. Stromelysin mRNA transcripts were localized to isolated individual cells, some of which were smooth muscle, in the plaque cap, intima, and adventitia, but not the media. Expression was also seen in large clusters of macrophages that contained intracellular lipid deposits. The isolated expression of stromelysin by smooth muscle cells may reflect local connective tissue remodeling associated with growth and the formation of the plaque, whereas the more extensive expression associated with macrophages may be of greater pathological significance, contributing to the destabilization of the extracellular matrix and eventual plaque rupture.

Arteriosclerosis↗