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Biomedical subjects

P Bentley

Publications and source records attributed to P Bentley.

At least 19 recordsLinked to original sources
PubMed1998

Flow cytometric assessment of three different methods for the measurement of in vitro apoptosis.

Chlorambucil-induced apoptosis was assessed by three different flow cytometric methods in B-cell chronic lymphocytic leukaemia (B-CLL) cells cultured in vitro and the results were compared with those derived from the morphological assessment of the same samples. Spontaneous apoptosis was consistently observed in the control cultures in the absence of drug but this accounted for less than 12% of all cells in every case. The methods under investigation were the Annexin V labelling assay, the terminal deoxynucleotidyl transferase (TdT) end-labelling assay and the labelling of a 38 kDa mitochondrial membrane protein (7A6 antigen) which is exposed on cells undergoing apoptotic cell death (Apo2.7 assay). The Annexin V assay consistently stained a higher percentage of cells and with a greater separation between the positive and negative cell populations. We conclude that the phosphatidyl serine translocation to the outer leaflet of the cell membrane following an apoptotic signal, as labelled by Annexin V, probably occurs before the development of the DNA strand breaks or the exposure of 7A6 antigen in those cells triggered to die by apoptosis.

Annexin A5↗
PubMed1998

Elevated Bcl-2/Bax are a consistent feature of apoptosis resistance in B-cell chronic lymphocytic leukaemia and are correlated with in vivo chemoresistance.

We investigated the relationship between drug resistance and Bcl-2/Bax in B-cell chronic lymphocytic leukaemia (B-CLL). Apoptosis was induced in vitro with chlorambucil and cell death was monitored by dual-labelled FACS analysis using Annexin V and propidium iodide. Bcl-2 and Bax protein expression was quantified using FACS and a correlation between drug-induced apoptosis and Bcl-2/Bax was established. Cells were then sorted into viable and nonviable populations according to their forward and side-scatter characteristics and re-analysed for Bcl-2/Bax. The most resistant cells had elevated Bcl-2 levels and low Bax expression. Furthermore, those cells which were undergoing apoptosis showed only a marginal reduction in Bcl-2 expression, but significantly elevated Bax expression following exposure to chlorambucil. The Bcl-2/Bax was significantly greater in the cell fractions resistant to chlorambucil-induced apoptosis. This observation further supports the suggestion that Bax is the pivotal protein in determining the fate of cells following apoptotic signals.

Antineoplastic Agents, Alkylating↗
PubMed1996

Failure to induce resistance to cytotoxic drugs in normal lymphocytes.

Lymphocytes from patients who had received chemotherapy for lung cancer were examined for evidence of drug resistance using an in vitro assay for sensitivity to the effects of chlorambucil and etoposide. There was no evidence of induced resistance to the effects of chlorambucil or etoposide in the patients' lymphocytes when compared to the sensitivity of the normal control subjects' lymphocytes. In four patients, in whose lymphocytes sensitivity to chlorambucil was measured before and after a course of treatment, there was no significant change in the in vitro drug sensitivity. These findings are in contrast to those made previously using the lymphocytes of patients with chronic lymphocytic leukaemia and indicate that acquired drug resistance is a property of malignant cells and is not inducible in normal lymphocytes.

Antineoplastic Agents↗
PubMed1996

Regulation of clinical chemoresistance by bcl-2 and bax oncoproteins in B-cell chronic lymphocytic leukaemia.

The bcl-2 gene was first shown to be dysregulated in the majority of follicular lymphomas in which a t(14;18) chromosomal translocation is present, but is also over-expressed in the absence of gene rearrangements in most cases of B-cell chronic lymphocytic leukaemia (B-CLL). The bcl-2 oncoprotein is a regulator of apoptosis and the activity of this protein is opposed by bax, a homologous protein that accelerates the rate of cell death. B-lymphocyte bcl-2 and bax protein levels were found to be significantly altered in B-CLL patients when compared to those of a normal control group. Increased bcl-2/bax ratios were observed in both the treated and untreated patients when compared to those of normal controls. These alterations were particularly pronounced in those treated patients found to be clinically unresponsive to chemotherapy.

Drug Resistance, Neoplasm↗
PubMed1996

Clinical audit in the National Health Service: fact or fiction?

It is increasingly recognized that the repeated rhetorical emphasis from 1989 to date on achieving measurable benefits to patients from audit, in the face of inattention to the development of methodologies with which to realize such benefits in operational practice, has represented a serious deficiency in strategic planning and direction and a consequent failure to establish functional clinical audit within the NHS. A grand revision of strategy is therefore necessary, and this should begin with the development of a research-based method of audit, the training of clinicians and audit support staff in its use and a subsequent trial of its effectiveness prior to its implementation within the NHS. Only then will measurable improvements become possible, value for money be assured and clinicians' attitudes to audit change.

Attitude of Health Personnel↗
PubMed1996

The Total Health Care Audit System: a systematic methodology for clinical practice evaluation and development in NHS provider organizations.

Writing in Medical Education in 1982, Fowkes (1982) noted the lack of general agreement within the medical profession on methods of audit, a deficiency previously articulated by Shaw (1980) and later emphasized by McIntyre (1985). More recently, a study by Black & Thompson (1993) of consultant and junior medical staff in four London district general hospitals revealed that 'many doctors did not understand how to undertake audit', and major research by both Hopkins (1993, 1994) and Buttery et al. (1994) described a multiplicity of methodological deficiencies in the general approaches to audit adopted by clinicians since the promulgation of the White Paper definition in 1989. Soundness of methodological approach is fundamental to securing the success of clinical audit within Provider organizations and is thus central to the generation of measurable improvements in the quality of clinical care being delivered to patients. It is therefore disturbing that methodological deficiencies may still be observed in general approaches to audit (Buttery et al. 1994), with no author yet recommending a formal system for critical inquiry into clinical practice. It was the recognition of the unsatisfactory nature of this situation which led us to develop a system aimed at assessing, in a critical fashion, the quality of the totality of care dispensed within NHS provider organizations. The system is presented here for the first time.

Algorithms↗
PubMed1995

The sensitivity of chronic lymphocytic leukaemia lymphocytes to irradiation in vitro.

The inhibition of [3H]-thymidine incorporation into the DNA of mitogen-stimulated chronic lymphocytic leukaemia lymphocytes by chlorambucil or gamma-irradiation in vitro was measured in a series of patients, some of whom were untreated, some treated and some who were showing resistance to first-line or second-line treatment. There was evidence of resistance to irradiation developing in parallel with that to chlorambucil. The resistance to chlorambucil in chronic lymphocytic leukaemia (CLL) is not necessarily due to altered drug transport or metabolism but to a more fundamental process affecting DNA damage.

Antineoplastic Agents, Alkylating↗
PubMed1994

Guinea-pig contact sensitization assays.

After reviewing general aspects and the historical development of predictive animal skin sensitization tests, the recommended tests of the OECD and EC are discussed. Background information is given on the changes which occurred in the last 2 years. From the many factors known to influence induction and challenge of contact allergy in animals, the discussion is focused on the choice of induction concentration and the vehicle. The difference of general screening strategies in Europe and the USA is presented and discussed. The paper closes with a discussion on the importance of inhouse validation of the methods used for proper prediction of the sensitizing potential of chemicals and with remarks on predictive in vitro tests.

Allergens↗
PubMed1994

Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine.

Ryanodine receptors/Ca2+ release channels play an important role in regulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work described here, we characterize the interaction of a tritium-labeled, photoactivable derivative of ryanodine (3H-labeled 10-O-[3-(4-azidobenzamido)propionyl]ryanodine ([3H]ABRy)) with the ryanodine receptor of skeletal, cardiac, and brain membranes. Scatchard analysis demonstrates that this ligand binds to a single class of high affinity sites in skeletal muscle triads. Furthermore, competition binding assays of [3H]ryanodine with skeletal, cardiac, and brain membranes in the presence of increasing concentrations of unlabeled ABRy illustrate that this azido derivative of ryanodine is able to specifically displace [3H]ryanodine from its binding site(s). Analysis of the effects of Ca2+, ATP, and KCl on [3H]ABRy binding in triad membranes shows a similar modulation of binding to that seen in these membranes with [3H]ryanodine. Photoaffinity labeling of triads with [3H]ABRy resulted in specific and covalent incorporation of [3H]ABRy into a 565-kDa protein that was shown to be the skeletal muscle ryanodine receptor. Digestion of the labeled ryanodine receptor revealed a [3H]ABRy-labeled 76-kDa tryptic fragment that was identified with an antibody directed against the COOH-terminal of the receptor. These results demonstrate that the 76-kDa COOH-terminal tryptic fragment contains the high affinity binding site for ryanodine.

Adenosine Triphosphate↗
PubMed1994

Radioimmunoassay for the calcium release channel agonist ryanodine.

A novel photo-activatable derivative of ryanodine, 9-hydroxy-21-(4-azidobenzoyloxy)-9-epiryanodine, has been synthesized and conjugated to keyhole limpet hemocyanin for the production of antibodies with high affinity and specificity to ryanodine. The anti-ryanodine antibodies reacted specifically on immunoblots with the azido-ryanodine compound covalently conjugated to bovine serum albumin. A radioimmunoassay specific for ryanodine was developed using the anti-ryanodine antibodies, and a dissociation constant for ryanodine of 1 nM was determined. Half-maximal inhibition constants (IC50) for various ryanodine derivatives were found to range between 3.2 and 200 nM. These IC50 values correlated very well with the IC50 values obtained for the compounds binding to the skeletal muscle membrane receptor. These antibodies should be useful for the characterization of the ryanodine binding site on the sarcoplasmic reticulum Ca2+ release channel.

Animals↗