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M J Wilson

Publications and source records attributed to M J Wilson.

At least 37 records · Page 2Linked to original sources
PubMed2001

Divergent and convergent evolution of NK-cell receptors.

Natural killer (NK)-cell receptors specific for major histocompatibility complex (MHC) class I molecules have been identified in humans and mice. Some of the most important receptors are structurally unrelated in the two species: the murine Ly-49 receptors are C-type lectins, while human killer-cell inhibitory receptors (KIRs) belong to the immunoglobulin superfamily. Here, Roland Barten and colleagues describe the divergent and convergent evolution of NK-cell receptors.

Animals↗
PubMed2001

The genomic context of natural killer receptor extended gene families.

The two sets of inhibitory and activating natural killer (NK) receptor genes belong either to the Ig or to the C-type lectin superfamilies. Both are extensive and diverse, comprising genes of varying degrees of relatedness, indicative of a process of iterative duplication. We have constructed gene maps to help understand how and when NK receptor genes developed and the nature of their polymorphism. A cluster of over 15 C-type lectin genes, the natural killer complex is located on human chromosome 12p13.1, syntenic with a region in mouse that borders multiple Ly49 loci. The equivalent locus in man is occupied by a single pseudogene, LY49L. The immunoglobulin superfamily of loci, the leukocyte receptor complex (LRC), on chromosome 19q13.4, contains many polymorphic killer cell immunoglobulin-like receptor (KIR) genes as well as multiple related sequences. These include immunoglobulin-like transcript (ILT) (or leukocyte immunoglobulin-like receptor genes), leukocyte-associated inhibitory receptor genes (LAIR), NKp46, Fc alphaR and the platelet glycoprotein receptor VI locus, which encodes a collagen-binding molecule. KIRs are expressed mostly on NK cells and some T cells. The other LRC loci are more widely expressed. Further centromeric of the LRC are sets of additional loci with weak sequence similarity to the KIRs, including the extensive CD66(CEA) and Siglec families. The LRC-syntenic region in mice contains no orthologues of KIRs. Some of the KIR genes are highly polymorphic in terms of sequence as well as for presence/absence of genes on different haplotypes. Some anchor loci, such as KIR2DL4, are present on most haplotypes. A few ILT loci, such as ILT5 and ILT8, are polymorphic, but only ILT6 exhibits presence/absence variation. This knowledge of the genomic organisation of the extensive NK superfamilies underpins efforts to understand the functions of the encoded NK receptor molecules. It leads to the conclusion that the functional homology of human KIR and mouse Ly49 genes arose by convergent evolution. NK receptor immunogenetics has interesting parallels with the major histocompatibility complex (MHC) in which some of the polymorphic genes are ligands for NK molecules. There are hints of an ancient genetic relationship between NK receptor genes and MHC-paralogous regions on chromosomes 1, 9 and 19. The picture that emerges from both complexes is of eternal evolutionary restlessness, presumably in response to resistance to disease.

Animals↗
PubMed2001

A molecular epidemiological study of sequential oral isolates of Candida albicans from terminally ill patients.

The pattern of candidal colonisation was studied in a group of terminally ill patients receiving antifungal treatment for oral candidosis. A total of 43 isolates of C. albicans was collected pre- and post-antifungal treatment from patients up to a maximum period of 4 weeks. Isolates were analysed by electrophoretic karyotyping (EK) and by inter-repeat polymerase chain reaction (IR-PCR). Fifteen electrophoretic karyotypes and 17 IR-PCR profiles were identified. Sequential isolates from 10 patients yielded identical profiles in both EKs and IR-PCR analyses. In the case of four patients, minor differences in the profiles were obtained by either EK or IR-PCR. The findings suggest that antifungal treatment in this patient group fails to eradicate the original C. albicans strain, thereby allowing recolonisation of the oral cavity. The present study has also shown that either EK or IR-PCR is a useful typing approach in such epidemiological investigations.

Antifungal Agents↗
PubMed2001

Strain persistence of invasive Candida albicans in chronic hyperplastic candidosis that underwent malignant change.

OBJECTIVES: The aim of this study was to assess persistence and tissue invasion of Candida albicans strains isolated from a 65 year-old patient with chronic hyperplastic candidosis (CHC), that subsequently developed into squamous cell carcinoma (SCC). MATERIALS AND METHODS: C. albicans (n=7) were recovered from the oral cavity of the patient over seven years. Confirmation of CHC and SCC in this patient was achieved by histopathological examination of incisional biopsy tissue. DNA fingerprinting was performed on the seven isolates from the CHC patient together with a further eight isolates from patients with normal oral mucosa (n=2), chronic atrophic candidosis (n=1), SCC (n=1) and CHC (n=4). Genotyping involved the use of inter-repeat PCR using the eukaryotic repeat primer 1251. Characterisation of the tissue invasive abilities of the isolates was achieved by infecting a commercially available reconstituted human oral epithelium (RHE; SkinEthic, Nice, France). After 24 h, C. albicans tissue invasion was assessed by histopathological examination. RESULTS: DNA fingerprinting demonstrated strain persistence of C. albicans in the CHC patient over a seven year period despite provision of systemic antifungal therapy. The strain of C. albicans isolated from this patient was categorised as a high invader within the RHE compared to other isolates. CONCLUSIONS: Candidal strain persistence was evident in a patient with CHC over seven years. This persistence may be due to incomplete eradication from the oral cavity following antifungal therapy or subsequent recolonisation from other body sites or separate exogenous sources. The demonstration of enhanced in vitro tissue invasion by this particular strain may, in part, explain the progression to carcinoma.

Aged↗
PubMed2001

Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa.

The expression of many virulence factors in Pseudomonas aeruginosa is dependent upon environmental conditions, including iron levels, oxygen, temperature, and osmolarity. The virulence of P. aeruginosa PAO1 is influenced by the iron- and oxygen-regulated gene encoding the alternative sigma factor PvdS, which is regulated through the ferric uptake regulator (Fur). We observed that overexpression of PvdS in strain PAO1 and a DeltapvdS::Gm mutant resulted in increased pyoverdine production and proteolytic activity compared to when PvdS was not overexpressed. To identify additional PvdS-regulated genes, we compared extracellular protein profiles from PAO1 and the DeltapvdS::Gm mutant grown under iron-deficient conditions. A protein present in culture supernatants from PAO1 but not in supernatants from DeltapvdS::Gm was investigated. Amino acid sequence analysis and examination of the genomic database of PAO1 revealed that the N terminus of this 27-kDa protein is identical to that of protease IV of P. aeruginosa strain PA103-29 and is homologous to an endoprotease produced by Lysobacter enzymogenes. In this study, the gene encoding an endoprotease was cloned from PAO1 and designated prpL (PvdS-regulated endoprotease, lysyl class). All (n = 41) but one of the strains of P. aeruginosa, including clinical and environmental isolates, examined carry prpL. Moreover, PrpL production among these strains was highly variable. Analysis of RNase protection assays identified the transcription initiation site of prpL and confirmed that its transcription is iron dependent. In the DeltapvdS::Gm mutant, the level of prpL transcription was iron independent and decreased relative to the level in PAO1. Furthermore, transcription of prpL was independent of PtxR, a PvdS-regulated protein. Finally, PrpL cleaves casein, lactoferrin, transferrin, elastin, and decorin and contributes to PAO1's ability to persist in a rat chronic pulmonary infection model .

Amino Acid Sequence↗
PubMed2001

Analysis of promoters recognized by PvdS, an extracytoplasmic-function sigma factor protein from Pseudomonas aeruginosa.

The alternative sigma factor PvdS is required by Pseudomonas aeruginosa for initiation of transcription from pyoverdine (pvd) promoters. Two divergent PvdS-dependent promoters (pvdE and pvdF) were characterized by deletion analysis, and the minimal promoter region for each included a sequence element, the iron starvation (IS) box, that is present in other pvd promoters. Site-directed mutagenesis showed that the IS box elements were essential for promoter activity in vivo. Band shift assays and in vitro transcription experiments showed that a complex of PvdS and core RNA polymerase required the presence of an IS box in order to bind to and initiate transcription from pvd promoters. These results indicate that IS box elements participate in sequence-specific recognition by PvdS to enable initiation of transcription from pvd promoters and are likely to represent a -35 sequence element for this sigma factor.

ATP-Binding Cassette Transporters↗
PubMed2001

PCR fingerprinting of Candida albicans associated with chronic hyperplastic candidosis and other oral conditions.

The purpose of this study was to genotype strains of Candida albicans to determine whether specific types were associated with chronic hyperplastic candidosis (CHC). A total of 67 candidal isolates from CHC patients (n = 17) and from patients with other oral conditions (n = 21) were genotyped by PCR fingerprinting employing two interrepeat primer combinations (1245 and 1246 primers or 1251 primer) and a single minisatellite-specific M13 primer. The most suitable primer for fingerprint analysis was found to be primer 1251, yielding well-resolved banding patterns. For the 67 isolates tested, PCR fingerprinting delineated 25 (1245 and 1246 primers), 27 (1251 primer), and 25 (M13 primer) profiles. The majority of C. albicans isolates from multiple sites within the mouth produced identical profiles (six out of nine subjects examined). For patients for whom a series of longitudinal isolates was available, strain persistence for up to 7 years was evident for five out of eight individuals, despite episodes of antifungal therapy. Computer-assisted comparison of the interrepeat PCR fingerprints identified seven distinct profiles that were shared among isolates from different individuals. However, no association was evident among isolates of C. albicans from specific clinical conditions. Eight isolates that were initially identified as C. albicans but having atypical PCR profiles were later confirmed as Candida dubliniensis. In conclusion, the genotypic data do not indicate clonal restriction of C. albicans with respect to CHC. Furthermore, these results have demonstrated that in the majority of individuals, colonizing populations of C. albicans are clonal in nature and exhibit strain persistence.

Candida albicans↗
PubMed2001

Identification of Candida dubliniensis, based on ribosomal DNA sequence analysis.

Differentiation of Candida albicans and the recently described C. dubliniensis has proven difficult due to the high degree of phenotypic similarity of these species. The present study examines sequence variations in the ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions of C. albicans (n = 5) and C. dubliniensis (n = 7) strains, with a view to identifying sequence differences that would enable consistent differentiation of these two species by restriction fragment length polymorphism (RFLP) analysis. The ITS1 and ITS2 regions, together with the entire 5.8S rRNA gene of the strains, were amplified by the polymerase chain reaction (PCR), using primers ITS1 and ITS4, PCR products from both C. albicans and C. dubliniensis were of similar size (around 540 bp); however, sequence analysis revealed over 20 consistent base differences between the products of the two species. On the basis of sequence variation, the restriction enzyme MspA1 I was selected and used to differentiate the PCR products of C. albicans and C. dubliniensis by RFLP analysis. MspA1 I yielded two discernible fragments from C. albicans PCR products, whilst those from C. dubliniensis appeared undigested, thereby providing an approach to differentiate the two species.

Candida↗
PubMed2000

Characterization of an ECF sigma factor protein from Pseudomonas aeruginosa.

The PvdS protein is essential for synthesis of the siderophore pyoverdine by Pseudomonas aeruginosa. PvdS has some sequence similarity to a family of alternative sigma factor proteins (the ECF [extracytoplasmic factor] family) that direct bacterial RNA polymerases to transcribe genes encoding extracytoplasmic factors. PvdS was purified as a His-tagged protein (hPvdS) and this was used to test the hypothesis that PvdS is a sigma factor protein. The purified protein caused core RNA polymerase from Escherichia coli to bind specifically to the promoters of pyoverdine synthesis genes and enabled transcription from these promoters in vitro. In addition, PvdS was found to co-purify with RNA polymerase from P. aeruginosa, indicating that PvdS associates with RNA polymerase inside the bacteria. These results show that PvdS is a sigma factor protein.

Bacterial Proteins↗
PubMed2000

Plasticity in the organization and sequences of human KIR/ILT gene families.

The approximately 1-Mb leukocyte receptor complex at 19q13.4 is a key polymorphic immunoregion containing all of the natural killer-receptor KIR and related ILT genes. When the organization of the leukocyte receptor complex was compared from two haplotypes, the gene content in the KIR region varied dramatically, with framework loci flanking regions of widely variable gene content. The ILT genes were more stable in number except for ILT6, which was present only in one haplotype. Analysis of Alu repeats and comparison of KIR gene sequences, which are over 90% identical, are consistent with a recent origin. KIR genesis was followed by extensive duplication/deletion as well as intergenic sequence exchange, reminiscent of MHC class I genes, which provide KIR ligands.

Alu Elements↗
PubMed2000

Arrangement of the ILT gene cluster: a common null allele of the ILT6 gene results from a 6.7-kbp deletion.

The leukocyte receptor cluster (LRC) is a highly polymorphic region of human chromosome 19q13.4 that encompasses at least 24 members of the immunoglobulin superfamily (Ig-SF). The centromeric end of the LRC contains eight Ig-SF loci, namely LAIR1 and seven ILT genes. All ILT genes conform to prototypic ILT gene structures. ILT6 is the only member of the ILT family that lacks a transmembrane and cytoplasmic domain. Close examination of the ILT6 genomic sequence reveals high similarity of this locus with the organization of activating ILT genes. However, the ILT6 transcript runs through the putative splice site of exon 8 that encodes for an extracellular stalk region, leading to a premature in-frame stop codon. Downstream of exon 8 are three pseudo exons that are not included in any of the known ILT6 transcripts, but share high homology to the equivalent region in activating ILT loci, suggesting that these genes have evolved from a common ancestral sequence. Comparison of two haplotypes over this region revealed a remarkable polymorphism with respect to the ILT6 gene which lacks exons 1-7 in one allele, reminiscent of the presence/absence variation displayed by the closely related and genetically linked KIR loci. Detailed sequence analysis of the two LAIR/ILT clusters suggests that the two complexes may have evolved from an inverted duplication.

Alleles↗
PubMed2000

Ascending aortic atheroma assessed intraoperatively by epiaortic and transesophageal echocardiography.

BACKGROUND: The presence of ascending aortic atheroma is a known risk for systemic emboli or early saphenous vein graft failure if unrecognized at the time of cardiopulmonary bypass. METHODS: This study prospectively compared intraoperative omniplane transesophageal echocardiography (TEE) and epiaortic ultrasound (EAU) images in 22 patients (6 women, 16 men, age 66 +/- 8 years) before surgical manipulation of the ascending aorta. Atheroma lesion severity was scored: 1 = normal, 2 = nonprotruding intimal thickening (> 2 mm), 3 = atheroma less than 4 mm +/- Ca++, 4 = atheroma greater than or equal to 4 mm +/- Ca++, and 5 = any size mobile or ulcerated lesion +/- Ca++. The ascending aorta between the aortic valve and innominate artery was divided into proximal, middle, and distal segments. A total of 66 segments were evaluated. RESULTS: Although the overall agreement of scores between procedures was 75.8%, significantly more lesions were identified by EAU (15) than by TEE (5) (p < 0.03). TEE failed to identify lesions in the middle and distal segments of the aorta with a score of more than 3. CONCLUSIONS: Although atheromatous lesions were identified in the ascending aorta by both ultrasound modalities, the results suggest that intraoperative EAU may have an advantage over TEE for surgeons assessing target sites for surgical procedures involving the ascending aorta.

Aged↗
PubMed2000

Down-regulation of inhibition following unilateral deafening.

Physiological and neurochemical experiments described here suggest that unilateral deafening causes a reduction in inhibition in the adult gerbil inferior colliculus (IC) contralateral to the deafened ear. Multiple-unit recordings were made from single electrode penetrations in the IC prior to and directly after contralateral cochlear ablation. These recordings showed up to 60% increases in the proportion of sampled loci at which neural activity excited by ipsilateral stimulation was observed after the ablation. Novel excitatory responses were evident within minutes of the ablation. Western blotting for glutamic acid decarboxylase protein levels showed significant decreases in the IC contralateral to cochlear ablation, relative to those in the ipsilateral IC, at 24 h and 7 days survival after the ablation. Four hour or 1 year survival post-ablation did not produce significant contralateral/ipsilateral differences in relation to the control group. Taken together, these results suggest the presence of at least two, short-term mechanisms involved in the central response to cochlear removal, both of which appear to implicate a decreased inhibitory influence. One is a very rapid, stimulus-related, functional unmasking. The other is a more delayed reduction in the capacity of gamma-aminobutyric acid synthesis in the IC.

Animals↗
PubMed2000

Glutathione loading prevents free radical injury in red blood cells after storage.

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1-6 degrees C for 0, 42 and 84 days in a conventional additive solution (Adsol) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4-6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (14C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (approximately 50%), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.

Biological Transport↗
PubMed2000

A comparison of performance of three gamma camera systems for positron emission tomography.

The development of coincidence positron imaging using a gamma camera should significantly increase PET utilization. This study has compared the performance of three such systems with each other and with an equivalent single photon emission computed tomography (SPECT) measurement. A Carlson phantom, which consisted of a 20 cm diameter cylinder containing areas of uniform activity, hot lesions, cold lesions and orthogonal alternate hot and cold lines, was filled with an activity of 18F chosen to try and best exploit each camera and imaged for a fixed time (27 min). The image quality of 9 mm thick slices in each section of the phantom was compared visually. Several image quality parameters were also compared including line source resolution and noise equivalent count rate. There were considerable differences in PET image quality between the three cameras but all were at least as good as the SPECT image. The full width at half maximum (FWHM) resolution of all systems was similar (approximately 4.5 mm) but the maximum noise equivalent count rates in a 20 cm cylinder were significantly different (6.3, 2.6 and 1.6 kcps) (where cps is counts per second) and correlated with the phantom image quality.

Fluorine Radioisotopes↗
PubMed2000

Phenotypic characterisation of Candida albicans isolated from chronic hyperplastic candidosis.

The phenotypes of 35 Candida albicans isolates from 19 patients with chronic hyperplastic candidosis (CHC) and 35 isolates from 30 patients with non-CHC infections were compared. Typing was based on carbohydrate assimilation, chemical sensitivity and serology. Eight carbohydrate assimilation profiles were evident with the API-20C system and a single profile predominated for isolates from CHC (17 of 19 patients; 89%) and non-CHC (18 of 30 patients; 63%). Chemical sensitivity tests revealed four profiles with no significant difference between CHC and non-CHC isolates. Serotype A predominated for isolates from both CHC (15 of 19 patients; 79%) and non-CHC (25 of 30 patients; 83%) infections. Boric acid resistance was more prevalent in CHC isolates, although a significant difference was not apparent. In summary, there was no overall difference in the phenotypes of isolates from CHC and non-CHC patients, and clonal restriction of CHC isolates was not demonstrated.

Candida albicans↗
PubMed2000

Evaluation of a phenotypic scheme for identification of the 'butyrate-producing' Peptostreptococcus species.

Gram-positive anaerobic cocci (GPAC) are isolated from approximately one quarter of all infections involving anaerobic bacteria. However, studies of the significance of this group of pathogens have been hindered by an inadequate taxonomy and the lack of a valid identification scheme. In the present study, a phenotypic scheme for the identification of 'butyrate-producing' GPAC based on the analysis of volatile fatty acid profiles by gas-liquid chromatography, biochemical profiles (including the use of the rapid ID 32 A commercial kit) and carbohydrate fermentation reactions, was evaluated. The identity of 68 clinical isolates of GPAC was determined by application of the scheme published by Murdoch. The scheme was found to be easy to apply and only four of the test isolates could not be readily assigned to a species or well-defined group. The species most frequently identified in the test collection were Peptostreptoccoccus vaginalis, P. tetradius and the betaGAL group. A large number of strains was assigned to the heterogeneous 'prevotii/tetradius' group. Some species regarded as being restricted to particular clinical sites were shown to be more widespread than previously thought. The clinical source of the isolates did not show any consistent correlation with species identity.

Butyrates↗