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Biomedical subjects

R J Watson

Publications and source records attributed to R J Watson.

At least 19 recordsLinked to original sources
PubMed2006

Targeting an E2F site in the mouse genome prevents promoter silencing in quiescent and post-mitotic cells.

Previous studies have shown that the cell cycle-regulated B-myb promoter contains a conserved E2F binding site that is critical for repressing transcription in quiescent cells. To investigate its significance for permanent promoter silencing, we have inactivated this binding site in the mouse genome. Mice homozygous for the mutant B-mybmE2F allele were fully viable, however, B-myb transcription was derepressed during quiescence in mouse embryo fibroblasts (MEFs) derived from mutant animals. Moreover, it was found that mutation of the E2F site resulted in abnormal maintenance of B-myb expression in senescent MEFs and in differentiated brain tissue. These findings therefore reveal a direct and primary role for repressive E2F complexes in silencing gene expression in post-mitotic cells. Analysis of histone modifications at the promoter showed that histone H3 lysine 9 was constitutively acetylated throughout the cell cycle in homozygous mutant MEFs. This mouse system is the first description of an E2F site mutation in situ and will facilitate the study of E2F function in vivo.

Animals↗
PubMed2003

Cell cycle regulation by the B-Myb transcription factor.

The expression of genes required for progression through the cell cycle is highly modulated through a regulatory axis containing the E2F transcription factor and retinoblastoma tumour suppressor protein families. One of the genes regulated through this mechanism encodes the B-Myb transcription factor, which has been shown to be critically required for early embryonal development in the mouse. Transcriptional activity of B-Myb is substantially enhanced in S phase through modification by cyclin A/cdk2, and the evidence points squarely to the major role being played by B-Myb during this phase of the cell cycle. We discuss in this review recent findings suggesting that B-Myb is a multifunctional protein that has, in addition to its transcriptional properties, the ability to interact directly with other regulators of the cell cycle.

Animals↗
PubMed2002

In vitro acoustic characterisation of four intravenous ultrasonic contrast agents at 30 MHz.

The acoustic properties of four ultrasonic contrast agents (Optison, Definity, SonoVue and Sonazoid) were studied at 30 MHz using a Boston Scientific ClearView Ultra intravascular ultrasound (US) scanner modified to allow access to the unprocessed US data. A range of contrast agent concentrations were studied using either saline or glucose as the diluent of choice. Mean backscatter power was measured over regions-of-interest (ROI) at distances of 1, 1.5, 2, 3, 4 and 5 mm from the centre of the intravascular probe and normalised to the US data collected from a standard glass reflector. For all of the agents, the mean backscatter power at 30 MHz varied in a linear manner with concentration between 0.01 million microbubbles/mL and 1 million microbubbles/mL. Furthermore, for two of the agents, mean backscatter enhancement was detectable at concentrations as low as 2 microbubbles/sample volume.

Albumins↗
PubMed2001

Inhibition of cyclin A/Cdk2 phosphorylation impairs B-Myb transactivation function without affecting interactions with DNA or the CBP coactivator.

Expression of the B-Myb transcription factor is directed by an E2F-dependent transcriptional mechanism to late G1 and S phases of the cell cycle, where its transactivation properties are enhanced post-translationally by cyclin A/Cdk2-mediated phosphorylation. Other experiments have shown that removal of the B-Myb C-terminus constitutively activates both transactivation and DNA-binding activities, suggesting that autoregulation by this inhibitory domain is counteracted by phosphorylation. We report here on further experiments to examine this hypothesis. The importance of this modification was first emphasized by showing that co-transfected dominant-negative Cdk2 (Cdk2DN) substantially reduced B-Myb transactivation activity. We then attempted to map the autoregulatory domain by analysing a series of progressively deleted C-terminal B-Myb mutants. Removal of just 29 C-terminal aa increased transactivation appreciably, however, maximal activity required removal of 143 amino acids (as in B-Myb + 561). Enhanced B-Myb + 561 function correlated with the acquisition of DNA binding activity to a single Myb binding site (MBS) oligonucleotide as determined by bandshift assays, however, further assays showed that even wt B-Myb could bind a DNA fragment containing three MBS. Although transactivation by B-Myb was severely dependent on hyperphosphorylation, neither inhibiting this activity by co-transfecting Cdk2DN nor augmenting it with cyclin A resulted in significant effects on DNA-binding. We also found that B-Myb could synergize with the CBP coactivator and that this cooperativity was cyclin A/Cdk2-dependent. Despite this, the physical association between these proteins was not influenced by the B-Myb phosphorylation status. We discuss these findings in relation to the autoregulation of B-Myb by the C-terminal domain.

Bone Neoplasms↗
PubMed2001

Three-dimensional forward-viewing intravascular ultrasound imaging of human arteries in vitro.

The aim of this work was to investigate the suitability of a novel forward-viewing intravascular ultrasound (IVUS) technique for three-dimensional imaging of severely stenosed or totally occluded vessels, where the conventional side-viewing IVUS systems are of limited use. A stiff 3.8 mm diameter forward-viewing catheter was manufactured to scan a 72 degrees sector ahead of its tip. Conical volume data were acquired by rotating the catheter over 180 degrees by means of a motorised mechanical system. Operating at 30 MHz, the catheter was integrated with an IVUS scanner and a radiofrequency data acquisition system. Postmortem carotid and femoral arteries were scanned in vitro. Correlation of the reconstructed images with histology demonstrated the ability of this forward-viewing IVUS system to visualise healthy lumens, bifurcations, thickened atherosclerotic walls and, most importantly, severe and complete vessel occlusions. A rotating-sector forward-viewing IVUS system is suitable for anatomical assessment of severely diseased vessels in three dimensions.

Arterial Occlusive Diseases↗
PubMed2001

Sinorhizobium meliloti cells require biotin and either cobalt or methionine for growth.

Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B(12)-dependent homocysteine methyltransferase for methionine biosynthesis.

Biotin↗
PubMed2001

Intact soil-core microcosms compared with multi-site field releases for pre-release testing of microbes in diverse soils and climates.

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

Agriculture↗
PubMed2000

Classification of arterial plaque by spectral analysis of in vitro radio frequency intravascular ultrasound data.

To test whether radio-frequency analysis of coronary plaques predicts the histological classification, r.f. data were collected using a 30 MHz intravascular ultrasound scanner. Two hundred ninety-nine regions-of-interest from eight postmortem coronary arteries were selected and identified by histology as falling into one of seven different tissue types. These are loose fibrous tissue (n = 78), moderate fibrous tissue (n = 27), dense fibrous tissue (n = 33), microcalcification (n = 14), calcified plaque (n = 55), lipid/fibrous mixture (n = 51) and homogeneous areas of lipid pool (n = 29). On the basis of a previous study, four spectral parameters were calculated for each of the regions-of-interest: maximum power (dB), mean power (dB), spectral slope (dB/MHz) over the bandwidth 18-35 MHz and the intercept of the spectral slope with the 0 Hz axis (dB). A minimum-distance classifier using the Mahalanobis (1948) distance was applied to the data. Following resubstitution of the training data into the classifier, the total correctly classified was 54%. The data were reclassified using three broader tissue groups: (1) calcified plaque, (2) lipid pool and (3) a mixed fibrous category, incorporating loose fibrous tissue, moderate fibrous tissue, dense fibrous tissue, lipid/fibrous mixture and microcalcification. The total correctly classified was 86%. Using "leave-one-out" cross-validation, the classification rates were 48% for seven tissue subgroups and 83% for three broader categories of tissue type.

Cadaver↗
PubMed2000

Purification and characterization of a common soil component which inhibits the polymerase chain reaction.

DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.

Canada↗
PubMed1999

Rapid dephosphorylation of p107 following UV irradiation.

In response to UV irradiation, mouse NIH3T3 fibroblasts transiently arrest predominantly in the G1 phase of the cell cycle. Here, we investigate the role of the retinoblastoma-related pocket proteins in this biological process. We report here that UV induces an increase in p107/E2F complexes, shown previously to be repressors of E2F-dependent transcriptional activity. Several lines of evidence indicate that the increase of p107/E2F complexes following UV irradiation is a consequence of rapid dephosphorylation of p107. First, UV-mediated p107 dephosphorylation could be abolished by pretreatment of NIH3T3 fibroblasts with the serine/threonine phosphatase inhibitors calyculin A and okadaic acid. Second, alteration of protein phosphatase 2A holoenzyme composition by over-expression of specific B subunits interfered with UV-mediated dephosphorylation of p107. Consistent with this, p107 could be dephosphorylated in vitro with PP2A. Moreover, dephosphorylation of p107 was shown to be independent of the activity of p53 and p21, as it occurred also in UV-treated p53-null as well as p21-null mouse fibroblasts. We observed a close correlation between the UV dosages required for G1 cell cycle arrest and p107 dephosphorylation. Our data suggest a model in which UV radiation-induced cell cycle arrest depends, at least in part, on the induction of a PP2A-like phosphatase that acts on p107.

3T3 Cells↗
PubMed1999

Accessing the literature on outcome studies in group psychotherapy: the sensitivity and precision of Medline and PsycINFO bibliographic database searching.

BACKGROUND: Bibliographic databases such as Medline and PsycINFO are major sources of initial information for systematic reviews. This study investigates the adequacy of Medline and PsycINFO in retrieving articles describing outcome studies of group-based psychological treatments. METHOD: Ten journals publishing a high number of group psychotherapy articles were hand searched for the publication years 1993 and 1994. They were then searched using narrow and broad strategies developed for each database. RESULTS: The sensitivities (number of relevant articles identified by each search divided by the total number of known relevant articles identified by all searches) of narrow and broad Medline searches were 28% and 67% respectively. Precision (the number of relevant articles identified by a search divided by the number of both relevant and irrelevant articles identified by the search) was 91% and 35% for narrow and broad searches respectively. The sensitivities of narrow and broad PsycINFO searches were 8% and 47% respectively. Precision was 30% for the narrow search and 36% for the broad search. The hand search identified 94% of all known articles. CONCLUSIONS: PsycINFO performed more poorly than Medline. Although it was possible to identify 67% of outcome studies using Medline, the low precision of the necessary search strategies would make it uneconomical in practice. Abstracts of papers not identified by either search strategy were examined for possible reasons for this failure. For Medline this was generally due to the terms used to identify group psychotherapy. For PsycINFO it appears that the indexing system does not allow the easy identification of outcome studies.

Databases, Bibliographic↗
PubMed1999

The use of stop signals to reduce the pain and distress of patients undergoing a stressful medical procedure: an exploratory clinical study.

The present study investigates stop signals and their effects on the pain and distress of patients undergoing a stressful medical procedure. Thirty-six chronic pain patients (17 men, 19 women) attending an out-patient operating theatre for diagnostic nerve blocks/local anaesthetic injections were allocated to one of two conditions (experimental and control). All patients received a standard information leaflet concerning the forthcoming injections. Additional information was given to those in the experimental group on four occasions (three orally, one written) before the injections which stated that they could halt the procedure at any time by saying 'stop'. Subjective measures of anxiety, pain, distress, sense of control over the procedure as well as observer ratings of patient distress and pain behaviour were obtained before, during and after the injections. After initial differences in pre-injection pain were controlled for the experimental group, patients rated themselves as less distressed during the injections and recorded lower state anxiety following treatment. In view of various methodological limitations of the present study its findings may only be accorded 'pilot study' status. These limitations are explored in the discussion along with their implications for a more robust replication study. Nevertheless the present findings provide tentative support for the hypothesis that the use of stop signals can reduce the stressful nature of diagnostic nerve blocks.

Adult↗
PubMed1999

Identifying randomized controlled trials of cognitive therapy for depression: comparing the efficiency of Embase, Medline and PsycINFO bibliographic databases.

This study sought to compare the sensitivity and precision of Embase, Medline and PsycINFO bibliographic database searches for randomized controlled trials of cognitive therapy for depression. Searches in each database combined with a hand search in five selected journals formed the total pool against which each search was assessed. Sensitivities of standard searches (index terms only) were 68%, 84% and 38% in Embase, Medline and PsycINFO respectively. Sensitivities of expert searches (index and free text terms) were 76%, 97% and 65% for Embase, Medline and PsycINFO respectively. Medline appears to be the most efficient at identifying articles describing psychological treatment evaluation.

Cognitive Behavioral Therapy↗
PubMed1998

The cell-cycle regulated transcription factor B-Myb is phosphorylated by cyclin A/Cdk2 at sites that enhance its transactivation properties.

Expression of the B-Myb transcription factor is upregulated during late G1 phase of the cell cycle by an E2F-dependent transcriptional mechanism. B-Myb is specifically phosphorylated during S phase, suggesting that a cyclin-dependent kinase (Cdk) regulates its activity. Consistent with this notion, the S phase-specific cyclin A/Cdk2 was found previously to enhance B-Myb transactivation activity in cotransfected cells. In this study we provide evidence that B-Myb is a direct physiological target for cyclin A/Cdk2. We demonstrate that B-Myb is an in vitro substrate for cyclin A/Cdk2, but not for cyclin D1/Cdk4 or cyclin E/Cdk2. By mutating candidate Cdk2 phosphorylation sites, we show that B-Myb is phosphorylated at Thr447, Thr490, Thr497 and Ser581 by cyclin A/Cdk2 in vitro and that these sites are also phosphorylated in cycling U-2 OS cells. Inhibition of endogenous Cdk2 by dominant negative Cdk2 attenuated phosphorylation of Thr447, Thr490 and Thr497, but had no effect upon Ser581 modification. B-Myb transactivation activity was significantly reduced in a mutant containing amino acid substitutions at all four identified cyclin A/Cdk2 sites and was constitutively low in Saos-2 cells where endogenous cyclin A/Cdk2 activity was unable to phosphorylate ectopically expressed B-Myb. These data indicate that phosphorylation by cyclin A/Cdk2 is directly involved in enhancing B-Myb transactivation activity and that levels of endogenous cyclin A/Cdk2 activity may contribute to cell line-specific B-Myb function.

Bone Neoplasms↗
PubMed1997

Activation of human B-MYB by cyclins.

B-MYB expression is associated with cell proliferation and recent studies have suggested that it promotes the S phase of mammalian cells. Based on its homology to the transcription factors c-MYB and A-MYB, B-MYB is thought to be involved in transcriptional regulation; however, its activity is not detectable in several cell lines. It was postulated that B-MYB function may depend on the presence of a cofactor, and recent studies suggested that B-MYB is phosphorylated specifically during S phase in murine fibroblasts. In this report we provide evidence that the product of the human B-myb gene can be activated in vivo by coexpression with cyclin A or cyclin E. Transfection studies showed that B-MYB was a weak transcriptional activator in SAOS-2 cells and was unable to promote their proliferation. In contrast, overexpression of both B-MYB and cyclin A or cyclin E caused a drastic increase in the number of SAOS-2 cells in S phase. Also, overexpression of cyclin A and cyclin E in SAOS-2 cells enhanced the ability of B-MYB, but not c-MYB, to transactivate various promoters, including the cdc2 promoter, the HIV-1-LTR, and the simian virus 40 minimal promoter. A direct role for cyclin-dependent activation of B-MYB was demonstrated using an in vitro transcription assay. These observations suggest that one mechanism by which cyclin A and E may promote the S phase is through modification and activation of B-MYB.

Cell Cycle↗