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Biomedical subjects

D J Hall

Publications and source records attributed to D J Hall.

At least 37 records · Page 2Linked to original sources
PubMed1999

An early S phase checkpoint is regulated by the E2F1 transcription factor.

The E2F1 transcription factor regulates transit of cells through the S phase checkpoint, dependent on its association with cyclin A/cdk2. Expression in cells of a mutant E2F1 lacking the cyclin A/cdk2 binding domain leads to partial arrest of cells at the S phase checkpoint. When subconfluent growing cells expressing this mutant E2F1 are analyzed in detail, it is shown here that they display a significantly reduced incorporation of 3H-thymidine into the DNA of each S phase cell, compared to control cells or to cells overexpressing full-length E2F1. Further, when cells are blocked at the G1/S phase border and released, there is a clear reduction in the amount of 3H-thymidine incorporated into the DNA of S phase cells by 1.5 hours post release. Considering a normal 6 hour S phase duration, the results show that the S phase checkpoint mediated by E2F1 is not a late S phase event but an early one.

3T3 Cells↗
PubMed1999

A novel MDMX transcript expressed in a variety of transformed cell lines encodes a truncated protein with potent p53 repressive activity.

The MDMX gene product is related to the MDM2 oncoprotein, both of which interact with the p53 tumor suppressor. We have identified a novel transcript of the MDMX gene that is expressed in a variety of cell lines, and in particular, in growing and transformed cells. This transcript is identical to the published sequence yet it has a short internal deletion of 68 base pairs. This deletion produces a shift in the reading frame after codon 114, resulting in the inclusion of a stop codon at amino acid residue 127 (full-length MDMX is 489 residues). This truncated MDMX protein is termed MDMX-S ("short form"), represents only the p53-binding domain, and appears to bind p53 better than full-length MDMX. The MDMX-S protein can be detected in cell extracts and when overexpressed is much more effective than MDMX at inhibiting p53-mediated transcriptional activation and induction of apoptosis. Since MDMX-S lacks the central and carboxyl-terminal regions contained within full-length MDMX, it is likely to play a key role in the regulation of cell proliferation and apoptosis in a way distinct from MDMX.

3T3 Cells↗
PubMed1999

Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae.

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.

Caspase 10↗
PubMed1999

Postmortem retrieval of total joint replacement components.

Improved understanding of the clinical outcomes of orthopedic implants requires the study of implants at the end of their service life. Previous studies focused on the retrieval of so-called failed implants during surgical revision. Interest is now moving to the study of successful implants, which are those still in place at the patient's death. A procedure was developed for recruitment of implant patients and their families and for safe and effective device and tissue retrieval after death. More than 50 retrievals were performed to support various research efforts.

Arthroplasty, Replacement↗
PubMed1999

Short segment fixation of thoracolumbar burst fractures without fusion.

There continues to be controversy surrounding the management of thoracolumbar burst fractures. Numerous methods of fixation have been described for this injury, but to our knowledge, spinal fusion has always been part of the stabilising procedure, whether this involves an anterior or a posterior approach. Apart from an earlier publication from this centre, there have been no reports on the use of internal fixation without fusion for this type of fracture. The aim of the study was to determine the outcome of patients with thoracolumbar burst fractures who were treated with short segment pedicle screw fixation without fusion. This is a retrospective review of 28 consecutive patients who had short segment pedicle screw fixation of thoracolumbar burst fractures without fusion performed between 1990 and 1993. All patients underwent a clinical and radiological assessment by an independent observer. Outcome was measured using the Low Back Outcome Score. The minimum follow-up period was 2 years (mean 3.1 years). Fifty percent of patients achieved an excellent result with the Low Back Outcome Score, while 12% were assessed as good, 20% fair and 16% obtained a poor result. The only significant factor affecting outcome was the influence of a compensation claim (P < 0.05). The implant failure rate (14% of patients) and the clinical outcome was similar to that from series where fusion had been performed in addition to pedicle screw fixation. The results of this study support the view that posterolateral bone grafting is not necessary when managing patients with thoracolumbar burst fractures by short segment pedicle screw fixation.

Adult↗
PubMed1999

An unusual case of urinary retention due to imperforate hymen.

A 15 year old girl presented to the accident and emergency (A&E) department with a 24 hour history of lower abdominal pain, and was found to have acute urinary retention. She was discovered to have an imperforate hymen with associated haematocolpos and haematometrium. This is rare and is hence a very unusual presentation to the A&E department. Patients presenting with retention of urine should be carefully assessed for the cause.

Adolescent↗
PubMed1999

Immediate psychiatric discharge letters by fax.

Much psychiatric care is provided outside the hospital setting. It is important for general practitioners (GPs) to have available information of good quality, provided promptly, after patients' discharges from in-patient psychiatric care to enable them to provide a high standard of follow-up care. In order to assess the value of hand-written Immediate Discharge Letters sent by fax we undertook a postal questionnaire survey of GPs, and examined a proportion of the clinical notes relating to 160 patients who between January and March 1998 had been discharged from in-patient care in the psychiatry admission wards at Crichton Royal Hospital, Dumfries. We found that structured letters, hand-written on a patient's discharge from in-patient status were generally valued by GPs as was their transmission by fax. Though certain deficiencies were confirmed in their completion, they are of value pending the arrival of a more definitive final discharge summary. We conclude that the continued use of such immediate discharge letters in psychiatry and their continued transmission by fax is justified.

Adult↗
PubMed1999

Tumor necrosis factor and gamma-interferon repress transcription from the c-myc P2 promoter by reducing E2F binding activity.

Transcription of the c-myc gene in HeLa cells has been shown to be repressed by the combined action of tumor necrosis factor (TNF) and gamma-interferon (gamma-INF). Shown here, these two cytokines inhibit proliferation of Hela cells with a coordinate inhibition of c-myc gene expression. It was found that these two cytokines exert their effects on the more proximal region of the c-myc P2 promoter. Using c-myc promoter:CAT constructs, it was found that the combined action of these cytokines significantly repress transcription from P2. This repression occurred through the E2F site within the promoter and not the ME1a2 or ME1a1 sites. However, these cytokines had no effect on transcription from the rous sarcoma virus promoter or the SV40 virus early promoter. Protein binding assays indicate that TNF and gamma-INF did not effect the ability of the ME1a2 factor to bind to its site but did significantly repress E2F factor binding to its DNA sequence.

Basic Helix-Loop-Helix Proteins↗
PubMed1998

Regulation of the human Na/K-ATPase beta1 gene promoter by mineralocorticoid and glucocorticoid receptors.

Expression of the human Na/K-ATPase beta1 subunit is regulated by a mineralocorticoid- and glucocorticoid-responsive elements. Here we identified an MR and GR responsive element, at positions -650 to -630, within the beta1 gene promoter that is required for both MR and GR activation. Independent expression of MR and GR activated by aldosterone or triamcinolone acetonide (TA) leads to significant transactivation of the beta1 promoter. Yet coexpression of both receptors activated by aldosterone plus TA or cortisol results in a much lower induction, indicating that coexpression of MR and GR is inhibitory. Gel shift mobility assay using an oligonucleotide including the 21-base pair MRE/GRE with whole cell extracts prepared from CV-1 cells overexpressing MR or GR showed specific MR and GR binding to this sequence. Additionally, antibodies to both MR and GR effectively supershifted the protein-DNA complexes, indicating that these receptors bound to the DNA sequence. Finally, the 21-base pair MRE/GRE was capable of activating transcription from a heterologous promoter in response to both aldosterone and TA. Together these data indicate that the 21-base pair sequence represents a true MRE/GRE and that optimal activation of the human Na/K-ATPase beta1 promoter is controlled by mineralocorticoid and glucocorticoid hormones. It appears that an interaction of MR with GR on the beta1 promoter effectively down-regulates transcription.

Aldosterone↗
PubMed1998

Increased camptothecin toxicity induced in mammalian cells expressing Saccharomyces cerevisiae DNA topoisomerase I.

The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin. This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug. The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells.

Animals↗
PubMed1998

Thirteen-year follow-up of deliberate self-harm, using linked data.

BACKGROUND: We describe a national cohort of individuals surviving an episode of deliberate self-harm (DSH). Subsequent admissions for DSH and mortality over the following 13 years were studied. METHOD: In 1981, 8304 individuals were discharged from Scottish general hospitals with a diagnosis of attempted suicide (E950-959). They were followed-up to the end of 1994 using the Scottish Linked Data Set. Mortality was compared to the Scottish population using person-years analysis. RESULTS: 2624 people (31.6%) were readmitted with further episodes of DSH. The median number of readmissions was 1, range 1-137. The observed:expected ratio for all-cause mortality was 2.26 (95% CI 2.13-2.26). One hundred and sixty-eight people (2%) died from suicide, and 46 (0.6%) from undetermined causes. The observed:expected ratio for suicide plus undetermined deaths was 12.17 (95% CI 10.64-13.91). Accidental deaths in men and homicide deaths in men and women were elevated. The pattern of deaths from other causes suggested that alcohol misuse was a contributory factor. CONCLUSIONS: People admitted to general hospitals in Scotland after attempted suicide are at high risk of readmission for further episodes of DSH. Long-term follow-up of such large cohorts is impractical, but services should review the scope for intervention in alcohol misuse following DSH.

Adolescent↗
PubMed1998

Cyclin A levels, the duration of S phase and sensitivity to a chemotherapeutic agent are altered in fibroblasts cultured on a fibronectin matrix.

Culture of murine embryonic fibroblasts, but not vascular smooth muscle cells, on a fibronectin matrix significantly shortens their transit time through the S phase of the cell cycle. This shortening corresponds to an increase in both cyclin A protein levels and active cyclin A/cdk2 complex. The increase in cyclin A protein appears due to a translational/post-translational mechanism since there is no increase in cyclin A mRNA following culture of the cells on fibronectin. Treatment of cells cultured on fibronectin with a short pulse of the S phase chemotherapeutic agent camptothecin, resulted in a relative protection from cell death when compared to cells cultured on tissue culture plastic. Thus, while the cells have increased rate of transit through S phase fibronectin-mediated signaling protects the cells from S phase mediated apoptosis. In addition, fibroblasts constitutively expressing a mutant E2F1 transcription factor (E2F1d87) have a lengthened S phase, due to a truncation of the cyclin A/cdk2 binding domain. Culture of these mutant- expressing cells on fibronectin did not shorten their S phase duration in spite of the fact that cyclin A levels and active cyclin A/cdk2 complex were significantly elevated. Thus, although the fibronectin signaling mechanisms culminating in elevated cyclin A were intact in these mutant E2F1 expressing cells, they were insensitive to the effects of this elevated cyclin A. The effect of the mutant E2F1d87 on slowing transit through S phase appears dominant over the effect of elevated cyclin A.

3T3 Cells↗
PubMed1998

A mutant E2F-1 transcription factor that affects the phenotype of NIH3T3 fibroblasts inefficiency associates with cyclin A-cdk2.

The amino-terminal domain of the E2F1 transcription factor is the site of association with cyclin A-cdk2, mapping to residues 87-94. A mutant of E2F1 lacking the first 87 amino acids (termed E2F1d87) has a number of potent effects on cellular phenotype when constitutively expressed in NIH3T3 fibroblasts. For example, in these fibroblasts the duration of S phase and the sensitivity to S phase chemotherapeutic agents are both increased. Since E2F1d87 only partially truncates the cyclin A-cdk2 binding domain, it was important to determine the level of cyclin A-cdk2 association with this mutant to correlate any reduction in association with the observed effects on the cell cycle. It was found that cyclin A-cdk2 binds E2F1d87 in an in vitro assay but that this binding is reduced approximately 8 fold compared with binding to full-length E2F1, whereas no detectable binding was seen to a mutant E2F1 that lacks the first 117 amino acids. Correspondingly, H1 kinase activity in E2F1d87 immunoprecipitates from E2F1d87-expressing cells was significantly reduced compared with that seen for full-length E2F1. From these data it appears that E2F1 with reduced cyclin A-cdk2 binding activity mediates the alteration in cell cycle parameters seen in these cells.

3T3 Cells↗
PubMed1997

Reduction in fibronectin expression and alteration in cell morphology are coincident in NIH3T3 cells expressing a mutant E2F1 transcription factor.

Fibronectin within the extracellular matrix plays a role in cell attachment, spreading, and shape, while it also affects aspects of cell proliferation. Transcription factors such as E2F1 are also known to regulate cell shape and cell proliferation. Yet, to date no linkage has been established between fibronectin expression and E2F1. We show here that cells constitutively expressing a mutant E2F1 protein (E2F1d87) produce reduced amounts of fibronectin mRNA and protein. The altered expression of fibronectin seen in the E2F1d87 expressing cells is due, in part, to a reduction in transcription from the fibronectin promoter. Providing exogenous fibronectin, but not Type I collagen or laminin, as a substrate for cell adhesion is sufficient to revert the altered morphology and reestablish actin-containing microfilaments lost in the mutant cell line. An additional characteristic of the cells expressing the mutant E2F1 is that they demonstrate slow growth and a doubling in S phase duration. While providing exogenous fibronectin as an adhesion substrate did not shorten the S phase duration in the mutant line, it did significantly shorten the S phase duration in the parental NIH3T3 cell line, implicating a role for the extracellular matrix in regulating S phase transit in normal cells.

3T3 Cells↗
PubMed1997

Multiprotein complex formation on the c-myc promoter.

The E2F element is a cis-acting DNA sequence within the P2 promoter of c-myc proto-oncogene. While it is required for optimal transcription, the multiprotein complexes formed on this site have not been well characterized. We show that in extracts of human glioblastoma cells and NIH3T3 fibroblasts, significant E2F transcription factor binding to the c-myc E2F site occurs as a both a monomer (the active form) and as only two mutually exclusive complexes with the retinoblastoma gene product (pRb) or the cyclin A protein. The E2F protein monomer was found predominantly in the cytosolic fraction of the cellular extracts while the pRb and cyclin A complexes in the nuclear fraction, indicating that the monomer has novel physical properties. Thus, protein complex formation on the c-myc E2F site appears to contribute in a unique way to transcriptional activation.

3T3 Cells↗
PubMed1997

Semen donor recruitment strategies--a non-payment based approach.

Actual and projected prohibition of payment for semen donation in the UK and Canada has increased the need to examine alternative methods of donor recruitment. Evidence from a number of sources suggests that there is a large group of current and potential donors who are motivated more by meeting esteem needs than by payment. We develop an argument for using social marketing tools to create systematically an esteem-based approach to donor recruitment as an alternative to the payment approach. We conclude that esteem is a useful method of reciprocating the gift that donors make.

Economics↗
PubMed1997

Evaluation of spatial resolution as a function of thickness for time-resolved optical imaging of highly scattering media.

Previous experimental and theoretical investigations of the utility of time-resolved methods as a means of optical imaging through the human breast have indicated that a spatial resolution of approximately 1 cm is achievable by isolating the shortest path length photons which propagate through the tissue. Studies have also shown that resolution may be improved further by extrapolating the measured distribution using an appropriate model of photon transport. The experiments described here were performed in order to observe the relationship between achievable spatial resolution and the thickness of the medium. For a given time gate, an improvement in the spatial resolution was observed as the object thickness was reduced. Overall, the results indicate that a breast compression of about 1 cm may improve the limiting spatial resolution by as much as 7 mm. Less encouraging is the implication that temporal extrapolation over several orders of magnitude in intensity is required to achieve a comparable improvement in spatial resolution.

Biophysical Phenomena↗