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Biomedical subjects

R Johnstone

Publications and source records attributed to R Johnstone.

At least 19 recordsLinked to original sources
PubMed2006

Functional dissociation of DeltaPsim and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis.

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Design of trials with dying patients: a feasibility study of cluster randomisation versus randomised consent.

There is little rigorous evidence to underpin clinical guidelines for palliative care. However, research in palliative care is difficult, especially with dying patients. Consent is a major issue, since staff do not wish to invite dying patients to participate in trials. We, therefore, conducted a feasibility study in two units within the North West Wales NHS Trust. We explored two novel approaches to research in palliative care -cluster randomisation and randomised consent. All patients admitted to the two units during the study were asked for permission to use their data for research. We allocated the two units, at random, to use cluster randomisation or randomised consent for three months, and then to crossover to the other design. Of 24 patients dying during cluster-randomised phases, 13 gave consent on admission to use their data and were, thus, eligible to enter the trial; however, defined eligibility criteria reduced these to six active participants. Of 29 patients dying during randomised consent phases, seven gave consent on admission to use their data; although two were eligible for randomisation, neither entered the trial. We judge that cluster randomisation is the more effective design for research with dying patients. Computer simulation, based on data from 1500 dying patients on the Welsh Integrated Care Pathway, shows that crossover cluster trials need much smaller samples than simple cluster trials. Furthermore, this study has shown that crossover cluster trials are entirely feasible. We recommend a 'definitive' trial to test the crossover design more widely.

Adult↗
PubMed2005

Adaptive management and its role in managing Great Barrier Reef water quality.

Adaptive management is the pathway to effective conservation, use and management of Australia's coastal catchments and waterways. While the concepts of adaptive management are not new, applications involving both assessment and management responses are indeed limited at the national and regional scales. This paper outlines the components of a systematic framework for linking scientific knowledge, existing tools, planning approaches and participatory processes to achieve healthy regional partnerships between community, industry, government agencies and science providers to overcome institutional barriers and uncoordinated monitoring. The framework developed by the Coastal CRC (www.coastal.crc.org.au/amf/amf/_index.htm) is hierarchical in the way it displays information to allow associated frameworks to be integrated, and represents a construct in which processes, information, decision tools and outcomes are brought together in a structured and transparent way for adaptive catchment and coastal management. This paper proposes how an adaptive management approach could be used to benefit the implementation of the Reef Water Quality Protection Plan (RWQPP).

Animals↗
PubMed2003

Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.

A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.

Isotopes↗
PubMed2002

T cell receptor usage in patients with non-progressing HIV infection.

It is still unclear why some patients with HIV progress more slowly than others to developing full blown AIDS. In this study using flow cytometry we have investigated the TCRBV repertoire of peripheral blood T lymphocytes in 17 long-term non-progressing HIV patients (LTNP) to determine if there is a biased usage of T cell receptor V gene products. Patients were identified from hospital records and entered into the study. Three colour flow cytometry was used to determine the expression of the TCRBV3S5, BV5S1, BV5S2, BV5S3, BV6S1, BV7S1, BV9, BV11, BV12, BV13, BV14, BV16, BV17, BV18, BV20, BV21S3, BV22, and BV23 by CD8 and CD4 positive cells isolated from the peripheral blood of patients and controls. Increases in the absolute numbers of CD8+ T cells expressing TCRBV2 and 8 were observed in the HIV-LTNP population (P < 0.05 in both cases). No differences were seen in numbers of CD8+ T cells expressing other TCRBV or in any TCRBV within the CD4+ T cell popu-lation. At follow up (1-2 years later), those patients in which CD4 levels were below 500 x 106/l were those initially found to have lower levels of TCRBV8 +ve CD8 cells. A significant increase in the absolute numbers of T cells coexpressing the gamma delta (gammadelta) T cell receptor and CD8 were also seen in the HIV-LTNP patients compared with controls (P = 0.002). The increase in CD8+ T cells in the HIV-LTNP patients may be interpreted as either an antigen specific, or group of antigen specific responses to viral antigen, or less likely a viral superantigen. A low level of TCRBV8, CD8+ T cells might be predictive of a more rapid disease progression and might indicate a protective role for this population in HIV infected patients. The increase in gammadeltaT cells bearing the CD8 coreceptor suggests a role for this cell type in the response to HIV infection.

Adult↗
PubMed2002

Use of autologous blood as part of the perfusate for cardiopulmonary bypass: a priming technique.

In an attempt to replace the oncotic and protein coating capabilities of serum albumin in the perfusate, we established a priming protocol that used autologous blood as part of the perfusate solution. Prior to March 1, 1999, our standard priming protocol was 1650 ml of crystalloid with 250 ml of 5% serum albumin and 5,000 units of heparin. After removing albumin from our prime, our standard protocol was altered to include 40 ml of the patient's autologous blood in 1,800 ml of crystalloid and 10,000 units of heparin. To determine the intraoperative effects of using albumin/crystalloid primes (Group A), autologous blood/crystalloid primes (Group B) and crystalloid primes (Group C), a total of 178 patients were sequentially evaluated. Intraoperative parameters evaluated were total protein (TP), colloid osmotic pressure (COP), platelets (Plts) and fluid requirements during cardiopulmonary bypass (CPB). During an overlapping 12-month period of time, 1,092 consecutive cardiac surgical cases using CPB (584 albumin prime; 508 autologous blood prime) were evaluated for clinical outcomes in terms of mortality and length of hospitalization. In addition, over a period of 15 months, 1,458 patients in both the autologous blood/crystalloid group and the crystalloid only group were evaluated for the incidence of high-pressure excursions (HPE) after going on bypass. Comparative reviews of TP, COP and Plts demonstrated no significant difference 10 min after the start of bypass between Groups A and B. However, in Group C, there was a statistically significant increase in the intraoperative fluid requirements during CPB, compared to both of the other groups. There was no significant difference in the incidence of HPE, with an occurrence of 1.04% in the crystalloid only group and 1.11% in the autologous blood/crystalloid group. Autologous blood perfusates were identical to albumin perfusates in their platelet protection and reduction of fluid shifts during the intraoperative period.

Aged↗
PubMed2001

The kinetics of the hypoferraemic response and changes in levels of alternative complement activity in diploid and triploid Atlantic salmon, following injection of lipopolysaccharide.

To study any possible effects of triploidy on the kinetics of the response of two non-specific disease factors, full sibling diploid and triploid Atlantic salmon were injected intraperitoneally with either lipopolysaccharide (1 mg kg(-1) body weight) or saline. Individually marked fish were repetitively blood sampled for up to 19 days. Total serum protein concentrations remained constant throughout the experiment indicating that the sampling regime did not cause haemodilution. The alternative complement pathway activity (measured by the titre of haemolytic activity against rabbit erythrocytes) in the serum of saline injected fish remained constant but in LPS-injected fish it fell to barely detectable levels 2 days after injection, but recovered to pre-treatment levels by about day 5. Triploid fish took slightly longer to reach full recovery levels than diploids. All groups of fish showed a hypoferraemic response, suggesting that the sampling regime was at least partially responsible. However, the response was more rapid and pronounced in the LPS-injected fish. In the latter, serum iron concentrations decreased to very low levels by day 2 post-injection in the diploid fish and by day 3 in the triploid fish. Pre-treatment iron levels were re-established by about 15 days post-injection in all groups. The data show only slight differences between the diploid and triploid fish, but the longer time taken for the triploids to recover complement activity and the slower onset of the hypoferraemic response following injection of LPS, suggest that they may be at a disadvantage compared with their diploid siblings in their defence against bacterial infections.

Animals↗
PubMed1999

Spatial and temporal trends of contaminants in Canadian Arctic freshwater and terrestrial ecosystems: a review.

The state of knowledge of contaminants in Canadian Arctic biota of the freshwater and terrestrial ecosystems has advanced enormously since the publication of the first major reviews by Lockhart et al. and Thomas et al. in The Science of the Total Environment in 1992. The most significant gains are new knowledge of spatial trends of organochlorines and heavy metal contaminants in terrestrial animals, such as caribou and mink, and in waterfowl, where no information was previously available. Spatial trends in freshwater fish have been broadened, especially in the Yukon, where contaminant measurements of, for example, organochlorines were previously non-existent. A review of contaminants data for fish from the Northwest Territories, Yukon and northern Quebec showed mercury as the one contaminant which consistently exceeds guideline limits for subsistence consumption or commercial sale. Lake trout and northern pike in the Canadian Shield lakes of the Northwest Territories and northern Quebec generally had the most elevated levels. Levels of other heavy metals were generally not elevated in fish. Toxaphene was the major organochlorine contaminant in all fish analyzed. The concentrations of organochlorine contaminants in fish appear to be a function not only of trophic level but of other aspects of the lake ecosystem. Among Arctic terrestrial mammals, PCBs and cadmium were the most prominent contaminants in the species analyzed. Relatively high levels (10-60 micrograms g-1) of cadmium were observed in kidney and liver of caribou from the Yukon, the Northwest Territories and northern Quebec, with concentrations in western herds being higher than in those from the east. For the organochlorine contaminants, a west to east increase in zigma PCBs, HCB and zigma HCH was found in caribou, probably as a result of the predominant west to east/north-east atmospheric circulation pattern which delivers these contaminants from industrialized regions of central and eastern North America to the Arctic via long-range atmospheric transport. Radiocesium contamination of lichens and caribou has continued to decrease. Significant contamination by PCBs and lead of soils and vascular plants was observed in the immediate vicinity and within a 20-km radius of DEW line sites in the Canadian Arctic. There was also evidence for transfer of PCBs from plants to lemmings. There was no evidence, however, that large mammals such as caribou living in the general area of the DEW line sites had elevated levels of PCBs. There is very limited temporal trend information for most contaminants in biota of Arctic terrestrial and freshwater environments.

Animals↗
PubMed1999

Muscle growth and development in normal-sex-ratio and all-female diploid and triploid Atlantic salmon.

Muscle development and growth were investigated in diploid populations of normal-sex-ratio and all-female Atlantic salmon (Salmo salar L.) and their triploid counterparts produced by high-pressure treatment. Somites were formed at the rate of 6 h-1 in both diploids and triploids at 6 degrees C. The rostral-to-caudal development of myotubes, myofibrils and acetylcholinesterase staining at the myosepta was slightly more advanced in triploid than in diploid fish, although the differences were smaller than among individual families. The c-met receptor tyrosine kinase was used as a molecular marker for the satellite cells involved in postembryonic muscle growth. Satellite cell nuclei comprised 17.5 % of total myonuclei in smolts and they were 24 % more abundant in diploid than in triploid fish. Cells expressing the myogenic regulatory factor myf-6, a marker of satellite cells committed to differentiation, represented 14.8 % of total myonuclei in diploids and 12.5 % in triploids. At ambient temperatures, the number of white muscle fibres in normal-sex-ratio fish increased more than 30-fold between the alevin and smolt stages, and approximately 3.5-fold further during the first year of seawater growth. The rate of muscle fibre recruitment in seawater stages was significantly greater in diploid than in triploid fish, reaching 1162 fibres day-1 and 608 fibres day-1, respectively, in all-female groups 800 days post-hatching. For 42 cm fork-length fish, there were approximately one-third more muscle fibres per myotome in diploid than in triploid groups, 649 878 and 413 619, respectively, for all-female fish. The probability density function of muscle fibre diameters in each fish was estimated using non-parametric smoothing techniques, and the mean densities for diploids (fD) and triploids (fT) were calculated. The peak fibre diameter was approximately 20 (micro)m in all age classes, irrespective of ploidy. Distinct bimodal distributions of muscle fibre diameter were evident in all groups 775 days and 839 days post-hatching, reflecting seasonal cycles of fibre recruitment. fD and fT were compared using a non-parametric bootstrap technique and the reference band representing the null-hypothesis indicated that there was no difference with ploidy. Reference bands for normal-sex-ratio fish at 315 days and 470 days indicated that diploids had a higher percentage of smaller-diameter fibres and that triploid distributions had a thicker right-hand tail. Similar differences in fD and fT of muscle fibre diameters were found for all-female fish, although the statistical evidence was less strong. Reference bands indicated differences in the middle range of the distributions of muscle fibre diameter in fish 620-775 days post-hatch, with triploids having a thicker right-hand tail. Thus, a lower density of satellite cells was associated with reduced rates of fibre recruitment but a compensatory increase in muscle fibre hypertrophy in triploid compared with diploid fish.

Animals↗
PubMed1997

The use of lidocaine in microvascular reconstruction.

Vasospasm is known to have an adverse effect on the survival of free tissue transfers. Prolonged vasoconstriction decreases blood flow to the flap and promotes thrombosis at the anastomotic site. Because of its wide availability and rapid effect, topically applied lidocaine is used by many surgeons to prevent and correct vasospasm. Mucosal absorption of lidocaine is known to be rapid. Absorption by the surgical bed during microvascular reconstruction has not previously been determined. We had three specific aims: (1) to determine whether systemic absorption occurs, (2) if so, to determine the degree of absorption, and (3) to determine whether the type of reconstruction affects the degree of absorption. Twelve consecutive patients were entered into the study, four in a myocutaneous reconstructive group and eight in a fasciocutaneous group. Baseline serum lidocaine levels were drawn. After the initial application of lidocaine, levels were drawn every 30 minutes. Final levels were drawn 120 minutes after the last application. The total amount of lidocaine used ranged from 720 mg to 2600 mg. Application ranged from 7.5 mg/minute to 34.7 mg/minute (2 to 9 times the estimated toxic dose of 4 mg/minute for a patient weighing 70 kg). Results ranged from undetectable (<0.5 microg/ml) to minimal therapeutic levels (1.6 microg/ml). Toxic levels (>6.0 microg/ml) were not encountered. Significant (p < 0.05) differences were found between the fasciocutaneous group, which generally had no detectable serum levels, and the myocutaneous group, which had detectable subtherapeutic to minimally therapeutic levels. Doses considered toxic by intravenous or mucous membrane application may safely be applied topically. Greater absorption was found in the myocutaneous group, which is believed to be a result of the richer absorption surface provided by the muscular tissue.

Absorption↗
PubMed1997

Expression and function of the leucine zipper protein Par-4 in apoptosis.

The prostate apoptosis response-4 (par-4) gene was identified by differential screening for genes that are upregulated when prostate cancer cells are induced to undergo apoptosis. The par-4 gene is induced by apoptotic signals but not by growth-arresting, necrotic, or growth-stimulatory signals. The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain at its carboxy terminus. We have recently shown that the Par-4 protein binds, via its leucine zipper domain, to the zinc finger domain of Wilms' tumor protein WT1 (R. W. Johnstone et al., Mol. Cell. Biol. 16:6945-6956, 1996). In experiments aimed at determining the functional role of par-4 in apoptosis, an antisense par-4 oligomer abrogated par-4 expression and activator-driven apoptosis in rat prostate cancer cell line AT-3, suggesting that par-4 is required for apoptosis in these cells. Consistent with a functional role for par-4 in apoptosis, ectopic overexpression of par-4 in prostate cancer cell line PC-3 and melanoma cell line A375-C6 conferred supersensitivity to apoptotic stimuli. Transfection studies with deletion mutants of Par-4 revealed that full-length Par-4, but not mutants that lacked the leucine zipper domain of Par-4, conferred enhanced sensitivity to apoptotic stimuli. Most importantly, ectopic coexpression of the leucine zipper domain of Par-4 inhibited the ability of Par-4 to enhance apoptosis. Finally, ectopic expression of WT1 attenuated apoptosis, and coexpression of Par-4 but not a leucine zipperless mutant of Par-4 rescued the cells from the antiapoptotic effect of WT1. These findings suggest that the leucine zipper domain is required for the Par-4 protein to function in apoptosis.

Amino Acid Sequence↗
PubMed1997

A novel quantitative immunoassay system for p53 using antibodies selected for optimum designation of p53 status.

AIM: To develop a highly sensitive and specific enzyme linked immunosorbent assay (ELISA) system for analysis of p53 protein in cancer lysates. METHODS: The anti-p53 monoclonal antibodies DO7, 1801, BP53.12, and 421, and anti-p53 polyclonal antiserum CM1 were assessed by immunohistochemistry and western blot analysis to identify those most suitable for determining p53 status of cancer cells. Antibodies with desired characteristics were used to develop a non-competitive sandwich type ELISA system for analysis of p53 expression in cancer cytosols. Using the ELISA, p53 protein concentrations were measured in a small series of breast cancers, and the quantitative values compared with p53 immunohistochemical data of the same cancers. RESULTS: DO7 and 1801 gave the most specific and reliable results on immunohistochemistry and western blot analysis. Using these two antibodies, a non-competitive sandwich type ELISA system was developed to analyse p53 quantitatively. Analysis of the breast cancer series showed a good correlation between immunohistochemistry and the ELISA-tumours were generally positive using both techniques. Discrepancies were noted however: some cancers were immunohistochemically negative but ELISA positive. One explanation for this may be that the ELISA is more sensitive than immunohistochemistry. CONCLUSION: The p53 ELISA system is a non-competitive double monoclonal antibody sandwich method, using DO7 and 1801 which have been shown to be highly specific for p53 protein by immunohistochemistry and western blot analysis. The lower threshold of the assay is 0.1 ng/ml analyte in an enriched recombinant p53 preparation. As p53 is now regarded as a protein associated with prognosis in breast and other cancers, the assay may have clinical applications.

Antibodies, Monoclonal↗
PubMed1995

Use of the biotinylated antibody DAKO-ER 1D5 to measure oestrogen receptor on cytokeratin positive cells obtained from primary breast cancer cells.

A method for the use of a biotinylated antibody (DAKO-ER 1D5) to quantify oestrogen receptors (ER) on tumour cells by flow cytometry is described. ER quantification was determined after treatment with saponin rendering cells permeable to ER antibody. Use of dual parameter labelling was performed utilizing a FITC-conjugated antibody (NCL-5D3) directed against cytokeratin 8/18. This allowed selection of breast cancer cells of epithelial origin by gating to exclude contaminating inflammatory and stromal cells. Use of such a gating technique was seen to identify cells with a higher level of ER expression. Using QC quantum bead standards, the number of ER binding sites per cell was assessed. Results were compared with conventional ER quantification using a radio-ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for ER quantification described is simple, rapid, and reproducible. The assay may be of particular value in measuring ER on urgent clinical samples. Advantages of this assay over the radio-ligand binding assay include reduction in use of radio-labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay.

Antibodies, Monoclonal↗
PubMed1995

A randomized controlled trial of a home exercise programme for elderly people with poor mobility.

Eighty-six elderly people with limited mobility and dependence in at least one activity of daily living were recruited to a home exercise study. The subjects (mean age 82 years) were allocated at random to either a strength exercise group, a mobility exercise group or a health education group. Subjects were visited for 30 minutes every 3-4 weeks by a physiotherapist who gave both verbal and written instruction. Sixty-nine of the original 86 completed the 6-month study, with five drop-outs from the strength group, ten drop-outs from the mobility group, and two drop-outs from the health education group. By the end of the study, there were no significant differences between the groups with regard to changes in outcome variables. The results showed a trend towards improvement in both the exercise groups in both Sit to Stand and Timed Get Up and Go tests, but this failed to attain statistical significance. Further work is required to identify the optimal exercise intervention for this subgroup of the elderly population.

Aged↗
PubMed1993

Distinct IgG1 and IgG3 subclass responses to two streptococcal protein antigens in man: analysis of antibodies to streptolysin O and M protein using standardized subclass-specific enzyme-linked immunosorbent assays.

The IgG subclass composition of antibodies to two streptococcal protein antigens in sera following infection was analysed by enzyme-linked immunosorbent assays (ELISA). The assays were standardized using 5-iodo-4-hydroxy-nitrophenacetyl (NIP)-specific chimeric antibodies, to permit quantitative comparisons between subclasses. Antibodies to streptolysin O (SLO) were predominantly IgG1, with only minor contributions from the other subclasses. In contrast, antibodies to M protein were distributed between the IgG1 and IgG3 subclasses, and in approximately half the sera IgG3 predominated. The ratio of IgG1:IgG3 was greater for SLO than for M protein in 22/23 sera. Little or no IgG4 antibody was detected to either antigen. Functional affinities of the IgG1 and IgG3 antibodies, determined by inhibition ELISA, were comparable for the two antigens. The demonstration that two protein antigens encountered during streptococcal infection elicit antibody responses with markedly different subclass profiles has implications for IgG subclass regulation and vaccine development.

Antibodies, Bacterial↗