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Biomedical subjects

R M Phillips

Publications and source records attributed to R M Phillips.

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

Correlation between myofilament response to Ca2+ and altered dynamics of contraction and relaxation in transgenic cardiac cells that express beta-tropomyosin.

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

Actin Cytoskeleton↗
PubMed1998

Angiogenesis in the hollow fiber tumor model influences drug delivery to tumor cells: implications for anticancer drug screening programs.

The National Cancer Institute uses the hollow fiber assay as part of its screening program for anticancer drug discovery. Angiogenesis to hollow fibers implanted s.c. has not been reported, thereby raising concerns about the efficiency of drug delivery and its subsequent effects on chemosensitivity. By extending postimplantation times beyond the 6-day period presently used, extensive vascular networks develop, resulting in both increased delivery and chemosensitivity to doxorubicin. This study suggests that present protocols used to evaluate compounds may produce false negative results, and additional studies to determine the predictive value of the assay are required.

Adenocarcinoma↗
PubMed1998

A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes.

Twenty-six laboratories used X and Y chromosome probes and the same procedures to process and examine 15,600 metaphases and 49,400 interphases from Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes. In Part I, each laboratory scored 50 metaphases and 200 interphases from a normal male and a normal female from its own practice. In Part II, each laboratory scored 50 metaphases and 200 interphases on slides prepared by a central laboratory from a normal male and a normal female and three mixtures of cells from the male and female. In Part III, each laboratory scored 50 metaphases (in samples of 5, 10, 15, and 20) and 100 interphases (in samples of 5, 10, 15, 20, and 50) on new, coded slides of the same specimens used in Part II. Metaphases from male specimens were scored as 98-99% XY with no XX cells, and 97-98% of interphases were scored as XY with 0.04% XX cells. Metaphases from female specimens were scored as 96-97% XX with 0.03% XY cells, and 94-96% of interphases were scored as XX with 0.05% XY cells. Considering the data as a model for any probe used with fluorescence in situ hybridization (FISH), a statistical approach assessing the impact of analytical sensitivity on the numbers of observations required to assay for potential mosaicisms and chimerisms is discussed. The workload associated with processing slides and scoring 50 metaphases and 200 interphases using FISH averaged 27.1 and 28.6 minutes, respectively. This study indicates that multiple laboratories can test/develop guidelines for the rapid, efficacious, and cost-effective integration of FISH into clinical service.

Cytogenetics↗
PubMed1998

Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours.

The poor blood supply to solid tumours introduces many factors that affect the outcome of chemotherapy, one of which is the problem of drug delivery to poorly vascularized regions of tumours. Whereas poor drug penetration has been recognized as a contributing factor to the poor response of many solid tumours, the question of drug penetration through multicell layers has not been thoroughly addressed, largely because of restrictions imposed upon these studies by the requirement for either radiolabelled or naturally fluorescent compounds. The aim of this study is to describe modifications made to a recently published assay that broadens the scope for assessing drug penetration during the early stages of drug development and to characterize the ability of various drugs to penetrate multicell layers. DLD-1 human colon carcinoma cells were cultured on Transwell-COL plastic inserts placed into 24-well culture plates so that a top and bottom chamber were established, the two chambers being separated by a microporous membrane. Drugs were added to the top chamber at doses equivalent to peak plasma concentrations in vivo and the rate of appearance of drugs in the bottom chamber determined by high-performance liquid chromatography (HPLC). Both 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine) and 7-[4'-(2-nitroimidazol-1-yl)-butyl]-theophylline (NITP) rapidly penetrated DLD-1 multicell layers (50.9 +/- 12.1 microm thick) with t(1/2) values of 1.36 and 2.38 h respectively, whereas the rate of penetration of 5-aziridino-3-hydroxymethyl-1-methyl-2-[1H-indole-4,7-dione] prop-beta-en-alpha-ol (EO9) and doxorubicin through multicell layers was significantly slower (t(1/2) = 4.62 and 13.1 h respectively). Inclusion of dicoumarol increases the rate of EO9 penetration, whereas reducing the oxygen tension to 5% causes a reduction in tirapazamine penetration through multicell layers, suggesting that the extent of drug metabolism is one factor that determines the rate at which drugs penetrate multicell layers. The fact that EO9 does not readily penetrate a multicell layer, in conjunction with its rapid elimination in vivo (t(1/2) < 10 min), suggests that EO9 is unlikely to penetrate more than a few microm from a blood vessel within its pharmacokinetic lifespan. These results suggest that the failure of EO9 in the clinic is due to a combination of poor drug penetration and rapid elimination in vivo.

Antibiotics, Antineoplastic↗
PubMed1998

Prospects for bioreductive drug development.

Bioreductive drugs are inactive prodrugs that are converted into potent cytotoxins under conditions of either low oxygen tension or in the presence of high levels of specific reductases. The biochemical basis for selectivity relies on the ability of oxygen to reverse the activation process and the presence of elevated reductase levels in some tumour types. Key criteria for an ideal bioreductive drug should include poor activity against aerobic cells, activation over a broad range of oxygen tensions and, penetration through the aerobic fraction of cells. In addition, the active drug should be capable of killing non-proliferating cells. Numerous compounds are currently at various stages of drug development but Mitomycin C, which is generally considered to be the prototype bioreductive drug, is the only one in clinical use today. Of the drugs currently being evaluated clinically, tirapazamine has definite clinical activity against a variety of solid tumours when used in combination with cisplatin. Other drugs, such as EO9 and various nitroimidazoles, have not been impressive in the clinic and further development is required to improve properties such as drug delivery in the case of indoloquinones. A novel approach to exploiting tumour hypoxia is the development of a gene-directed enzyme prodrug therapy (GDEPT) strategy, where a gene encoding for a prodrug activating enzyme has been placed under the control of a hypoxia responsive promoter sequence. It is generally recognised that bioreductive drugs must be directed towards patients whose tumours have hypoxic regions or have appropriate enzymological characteristics. In terms of identifying tumour hypoxia, there has been considerable progress in the development of nitroimidazole based hypoxia markers that can be detected either via non-invasive or invasive procedures. Another strategy currently undergoing preclinical evaluation is the use of agents that modulate tumour blood flow and synergistic effects have been reported between bioreductive drugs and photodynamic therapy or inhibitors of nitric oxide synthase for example. The development of clinically useful bioreductive drugs depends therefore on the expertise of scientists and clinicians with varying backgrounds. The purpose of this review is to describe and critically assess recent developments in this field, with particular emphasis being placed on drug development and strategies aimed at optimising bioreductive drug activity.

Journal Article↗
PubMed1998

5-substituted analogues of 3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]prop-2-en- 1-ol (EO9, NSC 382459) and their regioisomers as hypoxia-selective agents: structure-cytotoxicity in vitro.

A series of regioisomeric analogues of 3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]prop-2-en-1 -ol (EO9, NSC 382459) with the hydroxymethyl and hydroxypropenyl substituents situated at either the 2- or the 3-position of the indole ring were synthesized. The compound lacking the 2-hydroxypropenyl substituent (31) had similar properties to EO9 under both aerobic and hypoxic conditions against V79 cells and was more potent against a human tumour cell line (A549) than EO9. It was reduced by human DT-diaphorase (DTD) at more than double the rate of EO9, thus implicating the importance of the enzyme activation step. Compound 16 (lacking the 3-hydroxymethyl substituent) was a better substrate for human DTD than EO9, yet exhibited lesser toxicity under both aerobic and hypoxic conditions. The toxicity generated by 16 was attributed to the 5-aziridinyl moiety and suggests a greater contribution from the 3-substituent over the 2-substituent. The toxicity of EO9 was attributed to a combination of the aziridinyl group and the leaving group properties of the 3-hydroxymethyl substituent. In general, compounds with a 5-methylaziridinyl moiety, such as EO8, exhibited substantially better hypoxia-selectivity due to much slower reduction by DTD (20-fold), thus reducing aerobic potency. All compounds had similar electron affinities, as indicated by their one-electron reduction potentials.

Aerobiosis↗
PubMed1997

Influence of drug exposure parameters on the activity of paclitaxel in multicellular spheroids.

Paclitaxel is a chemotherapeutic drug which has clinical activity against several solid tumours including ovarian and metastatic breast cancers. Despite extensive preclinical evaluation in several experimental models, no studies have determined the effect of taxol on multicellular spheroids, a model which closely mimics the microregions of solid tumours. MCF-7 human breast carcinoma spheroids were significantly less sensitive than monolayers with IC50 values of 14.33 +/- 4.51 microM and 0.15 +/- 0.09 microM, respectively, following a 1 h drug exposure. Similarly, DLD-1 human colon carcinoma spheroids were also more resistant (IC50 = 33.0 +/- 8.89 microM) than monolayers (IC50 = 0.36 +/- 0.14 microM) following a 1 h drug exposure. Paclitaxel was unable to penetrate DLD-1 multicell layers (22 microns in thickness), suggesting that suboptimal drug exposures to paclitaxel occur in cells which reside some distance away from the surface of the spheroid. In the case of DLD-1 spheroids, extending the exposure time to 24 h whilst maintaining the same overall concentration x time (C x T) drug exposure parameters, resulted in greater cell kill (C x T required to kill 50% of cells = 13.67 +/- 3.21 microM/h) compared with 1 h drug exposures (C x T required to kill 50% of cells = 33.00 +/- 8.89 microM/h). Similar results were obtained with MCF-7 spheroids. In monolayers cultures, dose-response curves contained a marked plateau phase (a characteristic feature of cell cycle phase specific drug) and in the case of MCF-7 cells, cell kill was proportional to T as opposed to C x T. These results support the use of prolonged infusions of paclitaxel in the clinic, as extending the duration of drug exposure not only allows more cells to enter sensitive phases of the cell cycle, but would also allow paclitaxel more time to penetrate into avascular regions of solid tumours. It is likely that paclitaxel will only be effective against cells which reside close to tumour blood vessels and combination therapy with bioreductive drugs (such as tirapazamine) may produce synergistic effects in vivo.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DT-diaphorase).

NAD(P)H:quinone oxidoreductase (NQO1, EC 1.6.99.2) is an obligate two-electron reductase that can either bioactivate or detoxify quinones and has been proposed to play an important role in chemoprevention. We have previously characterized a homozygous point mutation in the BE human colon carcinoma cell line that leads to a loss of NQO1 activity. Sequence analysis showed that this mutation was at position 609 of the NQO1 cDNA, conferring a proline to serine substitution at position 187 of the NQO1 enzyme. Using polymerase chain reaction (PCR) analysis, we have found that the H596 human non-small-cell lung cancer (NSCLC) cell line has elevated NQO1 mRNA, but no detectable enzyme activity. Sequencing of the coding region of NQO1 from the H596 cells showed the presence of the identical homozygous point mutation present in the BE cell line. Expression and purification of recombinant wild-type and mutant protein from E. coli showed that mutant protein could be detected using immunoblot analysis and had 2% of the enzymatic activity of the wild-type protein. PCR and Northern blot analysis showed moderate to low levels of expression of the correctly sized transcript in the mutant cells. Immunoblot analysis also revealed that recombinant mutant protein was immunoreactive; however, the mutant protein was not detected in the cytosol of either BE or H596 cells, suggesting that the mutant proteins were either not translated or were rapidly degraded. The absence of any detectable, active protein, therefore, appears to be responsible for the lack of NQO1 activity in cells homozygous for the mutation. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for the mutation at position 609 conducted on 90 human lung tissue samples (45 matched sets of tumour and uninvolved tissue) revealed a 7% incidence of individuals homozygous for the mutation, and 42% heterozygous for the mutation. These data suggest that the mutation at position 609 represents a polymorphism in an important xenobiotic metabolizing enzyme, which has implications for cancer therapy, chemoprevention and chemoprotection.

Adult↗
PubMed1997

Plateau-phase cultures: an experimental model for identifying drugs which are bioactivated within the microenvironment of solid tumours.

A commonly used technique for evaluating potential bioreductive drugs is the determination of hypoxic cytotoxicity ratios in vitro. This experimental model, however, does not accurately mimic the tumour microenvironment, as other factors (such as reduced pH, poor nutrient status, low cell proliferation rates and high catabolite concentrations) are not incorporated into the design of the assay. Plateau-phase monolayer cultures possess many of these characteristics, and this study compared the response of plateau-phase and exponentially growing human colon carcinoma cells (DLD-1) with a series of standard and bioreductive compounds. All drugs tested were added directly to conditioned medium and three patterns of chemosensitivity were observed. In the case of doxorubicin, vinblastine and 5-fluorouracil, exponentially growing cells were significantly more responsive than plateau-phase cultures. ThioTEPA and MeDZQ (2,5-diaziridinyl-1, 4-benzoquinone) were equally cytotoxic to both populations of cells. Tirapazamine (SR4233), RSU 1069, mitomycin C and EO-9, however, were preferentially toxic towards plateau-phase compared with exponentially growing cells. While the exact mechanisms responsible for these observations in each case are not known, this study suggests that plateau-phase cultures may prove to be a useful experimental model in the evaluation of drugs designed to work preferentially within the tumour microenvironment.

Antineoplastic Agents↗
PubMed1996

Bioreductive activation of a series of analogues of 5-aziridinyl-3-hydroxymethyl-1-methyl-2-[1H-indole-4, 7-dione] prop-beta-en-alpha-ol (EO9) by human DT-diaphorase.

The enzyme DT-diaphorase (NAD(P)H:quinone acceptor oxidoreductase, EC 1.6.99.2.; DTD) is believed to be a good target for enzyme-directed bioreductive drug development because elevated levels of enzyme activity have been described in several human tumour types and it plays a key role in the bioreductive activation of several quinone-based anticancer drugs. As part of an ongoing program to develop new bioreductive drugs, the ability of a series of indoloquinone compounds to serve as substrates for and to be bioreductively activated by purified recombinant human DTD was investigated. Of the seven compounds evaluated, EO9, EO68 and EO4 were substrates for human DTD, but only EO4 was reduced to a DNA cross-linking species, and this DNA damage was both concentration dependent and inhibited by dicoumarol. A broad spectrum of chemosensitivity was observed in the H460 non-small cell lung cancer cell line, with the most potent compounds being EO4 (IC50 = 23.9 nM), EO9 (IC50 = 34.5 nM) and EO68 (IC50 = 37.8 nM). Relatively minor structural changes resulted in major changes in both substrate specificity and cytotoxic potency. Comparative chemosensitivity studies demonstrated that EO4, EO9 and EO68 are preferentially toxic towards DTD-rich H460 cells compared with DTD-deficient H596 cells (ratio of IC50 values for H596 cells to H460 cells were 113.8, 92.2 and 103.9 respectively). In conclusion, this study has identified two new compounds that are substrates for human DTD, one of which (EO4) is reduced to a DNA cross-linking species. Further studies in a broad panel of cell lines and human tumour xenografts are warranted for EO4 and EO68 based upon the result of this study.

Antineoplastic Agents↗
PubMed1996

2,3-Butanedione 2-monoxime (BDM) induces calcium release from canine cardiac sarcoplasmic reticulum.

2,3-Butanedione 2-monoxime (BDM) is a well known inhibitor of skeletal and cardiac muscle contraction. Recently, it has been discovered that BDM has an influence on the sarcoplasmic reticulum (SR). We investigated the effects of BDM on the SR in our digitonin lysed myocyte system, which measures accumulated SR Ca2+. While BDM (30 mM) had no effect on SR Ca2+ uptake (under conditions that included Ca2+ release channel efflux inhibitors), it induced SR Ca2+ release (no efflux inhibitors) with a maximal reduction of 72% of SR Ca2+ at pCa 6.0. A titration showed that even 5 mM BDM resulted in a 45% reduction at that same pCa. Also, a positive correlation was found between the degree of BDM induced Ca2+ release and free Ca2+ concentration. Thus, the use of even low concentrations of BDM as an excitation-contraction uncoupler must be approached with caution.

Animals↗
PubMed1996

Synthesis and antitumour activity of new derivatives of flavone-8-acetic acid (FAA). Part 1: 6-Methyl derivatives.

A range of 17 derivatives of flavone-8-acetic acid (FAA) with a 6-methyl substituent have been prepared and their anti-tumour activity evaluated in vitro against a panel of human and murine tumour cell lines and in vivo against MAC 15A. While many of the compounds show activity comparable to FAA in vitro, this essentially disappears in vivo, possibly due to degradation before the compounds can reach the tumour site.

Animals↗
PubMed1996

The practitioner-teacher: a study in the introduction of mentors in the preregistration nurse education programme in Wales: Part 2.

This paper is the second of two which together report on a 2-year research project (Davies et al. 1994) investigating the implementation and impact of introducing mentors in the Common Foundation Programme of the new scheme for preregistration nurse education. The study was commissioned by the Department of Health and undertaken on an all-Wales basis. The first paper (Phillips et al. 1996) set the scene by discussing some underlying issues to policy reforms in nurse education, then placing the study setting within the context of a changing scenario in the National Health Service. The key techniques of data collection were then outlined (interviews, diary accounts, questionnaires, observations in clinical settings). This second paper will present the key themes to emerge from the data and suggest some important issues which arise from the findings of the research, worthy of further consideration and debate.

Education, Nursing↗
PubMed1996

The practitioner-teacher: a study in the introduction of mentors in the preregistration nurse education programme in Wales: Part 1.

This study focused on the introduction of mentors in the Common Foundation Programme of Project 2000 schemes of preregistration nurse education. The research, which was commissioned by the Department of Health Research and Development Division on behalf of the Welsh Office Nursing Division, began in February 1992 and was undertaken on an all-Wales basis. The completed report aims to provide policy makers with information relating to important issues which are central to the teaching and learning of nursing in clinical locations. This first paper discusses some background issues and gives a brief conceptual framework for considering policy reform, summarizes the research questions which emerged and describes the methods used to address them. The second paper (to appear in the next issue of the journal) will describe the key findings from the study and discuss some ensuing potential implications and considerations for all those involved in the preparation of future practitioners of nursing.

Clinical Competence↗
PubMed1996

Ultrasound protocol and quality control procedures in the European Lacidipine Study on Atherosclerosis (ELSA).

The European Lacidipine Study on Atherosclerosis (ELSA) is a multinational interventional clinical trial aimed at determining the antiatherosclerotic effects of Lacidipine, a calcium antagonist, when compared to atenolol, a beta-blocker, on the carotid arteries of 2300 cardiovascular asymptomatic patients with moderately high blood pressure. Quantitative B-mode ultrasound imaging is being used to measure the intima-media thickness of a standardized section of the carotid arteries including the distal common, bifurcation, and proximal internal carotids. Prospective investigations of large samples of population using ultrasonographic endpoints rely heavily on the precision and reproducibility of the method. Therefore, specific quality control protocols are required to determine and monitor cross-sectional and longitudinal stability of the measurement reproducibility. In ELSA, the ultrasound methodology was specifically designed to include a set of procedures to quality control the critical components of measurement variation including instrumentation, and ultrasound operators, i.e. sonographers and readers. The ELSA clinical trial will provide the largest set of prospective quality control data on the use of quantitative B-mode ultrasound imaging.

Arteriosclerosis↗
PubMed1995

Vaccination of racing greyhounds: effects on humoral and cellular immunity.

Greyhound kennel owners frequently employ multiple vaccination schedules in an attempt to reduce financial losses incurred as a result of infectious diseases. In order to determine the effects of multiple vaccination schedules on the immune system of racing greyhounds, three litters of greyhound pups raised in laboratory conditions were divided into two groups and subjected to either a maximum or a minimum vaccination schedule. Blood samples were collected biweekly for 6 months beginning at 2 weeks of age and analyzed to establish 'baseline' values for the lymphatic system of greyhounds. Lymphocyte transformation, total and differential leukocyte counts, and flow cytometry were used to evaluate cellular immunity. Humoral immunity was evaluated using serum neutralization and hemagglutination inhibition tests. Proliferation of peripheral blood lymphocytes in response to the mitogen concanavalin A (Con A) was higher for the maximum vaccination groups. The frequency distribution of circulating CD4 and IgG labeled lymphocytes was higher in the minimum vaccination groups. A significant treatment by time interaction in CD4, IgG, and IgM labeled cells was observed, This interaction, however, was not significant at any point in time for CD4 and IgG labeled cells. The percentage of lymphocytes expressing surface IgM was significantly higher in the minimum vaccination groups at 10 and 14 weeks of age. No significant differences were detected in humoral immunity between the maximum and minimum groups of each litter. Results of this study indicate that maximum vaccination schedules do not appear to be more effective or more immunosuppressive than minimum vaccination schedules.

Animals↗
PubMed1995

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.

Animals↗