Search PubMed⌕ Search

Biomedical subjects

C J McNeil

Publications and source records attributed to C J McNeil.

At least 37 records · Page 2Linked to original sources
PubMed1996

Tyrosinase may protect human melanocytes from the cytotoxic effects of the superoxide anion.

Reactive oxygen species (ROS) are potentially cytotoxic and several mechanisms have evolved to protect against their damaging effects. In melanocytes, tyrosinase may have such a rôle by utilising the superoxide anion (O2-) in the production of melanin. In the present study, we have examined the cytotoxic effects of O2- and hydrogen peroxide (H2O2) in human melanocytes both before and following the activation of tyrosinase. Xanthine oxidase (XO, 5-150 mU.ml-1) and glucose oxidase (GO, 0.1-20 mU.ml-1) were used to generate the O2- and H2O2 respectively, and the cytotoxic effects assessed by measuring cell survival using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay. 3 h later, dose-related decreases in melanocyte survival were seen. Similar experiments with keratinocytes and fibroblasts showed that these cells were more resistant to the cytotoxic effects of O2- than were the melanocytes. The effect of increasing tyrosinase activity was examined by growing the melanocytes in the presence of an analogue of melanocyte-stimulating hormone (MSH) Nle4DPhe7 alpha-MSH (10(-8) M), for 48 h. This increased tyrosinase activity, melanin content, the ability to trap O2- and the resistance of the melanocytes to the cytotoxic effects of this ROS, but failed to alter their susceptibility to the damaging effects of H2O2.Nle4DPhe7 alpha-MSH had no effect on the resistance of keratinocytes and fibroblasts to either O2- or H2O2. After 3 h, XO, as opposed to GO, also increased the melanin content of human melanocytes; this effect was not accompanied by an increase in tyrosinase activity. The present results suggest that tyrosinase may utilise O2- to produce melanin and that this process may protect melanocytes from the potentially damaging effects of this ROS.

Cell Survival↗
PubMed1996

Activation of tyrosinase reduces the cytotoxic effects of the superoxide anion in B16 mouse melanoma cells.

Tyrosinase may protect against oxidative stress by using the superoxide anion (O2-1.) in the production of melanin. We have examined this by comparing its cytotoxic effects in B16/F10 and B16/F10-differential deficient (-DD) mouse melanoma cells that express high and low levels of tyrosinase activity respectively. Xanthine oxidase (XO) was used to generate O2.1 and cytotoxicity assessed by measuring cell survival. XO increased O2.- concentrations and 3 h later dose related decreases in cell survival were seen. F10 cells were more resistant to these cytotoxic effects than the F10-DD cells. [Nle4, DPhe7]MSH increased tyrosinase activity and melanin content, reduced O2.- concentration and increased the resistance of F10 cells to the cytotoxic effects of O2.-. No such effects were seen in F10-DD cells. The effect of [Nle4, DPhe7]MSH on the resistance of the F10 cells was time-dependent and noticeable when tyrosinase activity but not melanin was increased. This suggests that it was the activation of tyrosinase rather than the increase in the melanin that provided the protection against O2.-. In support of this, inhibition of tyrosinase with phenylthiocarbamide reduced the increased resistance induced by [Nle4, DPhe7]MSH. Moreover, although melanin was capable of scavenging O2.- it had little effect at concentrations comparable to those in the activated F10 cells. XO also increased the melanin content of F10 but not F10-DD cells. We conclude that tyrosinase is able to utilise O2.- to produce melanin and this provides pigment cells with a unique anti-oxidant mechanism.

Animals↗
PubMed1996

Parathyroid hormone induces superoxide anion burst in the osteoclast: evidence for the direct instantaneous activation of the osteoclast by the hormone.

We have shown that superoxide anion (O2-) production by the osteoclast can be used as an index of the osteoclast activity since the agents that inhibit and stimulate the osteoclast also diminish and stimulate O2- production respectively. Therefore, we have investigated the mechanism of parathyroid hormone (PTH)-mediated stimulation of osteoclast function in terms of its effect on O2- generation. The determination of O2- generation was carried out by employing cytochrome c immobilised on a surface-modified gold electrode. The basal level of free radical production by the osteoblast-like cells (ROS 17/2.8) was 10(4)-fold lower than by osteoclasts cultured on bone. PTH had no acute effect on free radical production by the osteoblasts. The exposure of the osteoclasts cultured on bone to PTH led to a dramatic and immediate stimulation of O2- generation which was unaffected by the presence of ROS 17/2.8 cells. The osteoclasts co-cultured with ROS 17/2.8 cells and exposed to PTH for 3 h were also found to produce greater stimulation of O2- than the osteoclasts exposed to PTH alone. A competitive leukotriene D4 antagonist REV 5901, which also inhibits 5-lipoxygenase, did not block O2- generation by osteoclasts cultured alone or in the presence of osteoblasts. Therefore, we conclude that PTH directly stimulates osteoclasts to produce O2-; this may be the main mode of activation of the osteoclasts, although an osteoblast-mediated effect of the hormone cannot be ruled out.

Animals↗
PubMed1995

Direct electron transfer bioelectronic interfaces: application to clinical analysis.

Bioelectronic interfaces based on direct electron transfer to proteins and enzymes immobilised at functional electrode surfaces are currently under development and the potential of two such systems for application to clinical measurement will be outlined. The first is the detection of free radical production via direct electrochemistry of cytochrome c immobilised covalently at modified gold electrodes. The redox protein cytochrome c has been immobilised covalently to gold electrodes surface-modified with N-acetyl cysteine via carbodiimide condensation. The electrodes thus produced were used to measure directly the enzymatic and cellular production of the superoxide anion radical (O2(-). The superoxide radical reduced the immobilised cytochrome c which was immediately re-oxidised by the surface-modified gold electrode poised at a potential of +25 mV (vs Ag/AgCl). The electron transfer rate constant (ket) of this process was 3.4 +/- 1.2 s(-1). The rate of current generation was directly proportional to the rate of O2(-) production. The essentially reagentless system produced was designed to be applied ultimately to continuous monitoring of free radical activity in vivo since there is evidence that oxygen-derived free radical species act as mediators which cause and perpetuate inflammation in disease states, including rheumatoid arthritis and neurodegenerative disorders. The second systems are pseudo-homogeneous immunoassays based on direct electron transfer to horseradish peroxidase. Horseradish peroxidase enzyme electrodes based on activated carbon (HRP-ACE) have been constructed by simple passive adsorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome c Group↗
PubMed1995

Specific binding assay for biotin based on enzyme channelling with direct electron transfer electrochemical detection using horseradish peroxidase.

A model 'homogeneous' format enzyme channelling specific binding assay for biotin based on peroxide-sensitive horseradish peroxidase mediatorless enzyme electrodes is described. The procedure involved the immobilisation of avidin onto the surface of printed carbon horseradish peroxidase (HRP) enzyme electrodes and the competitive binding of biotin and biotinylated glucose oxidase. Upon addition of glucose, hydrogen peroxide was generated via the glucose oxidase label. Direct electron transfer between the electrodes and HRP resulted in the detection of H2O2 by electroenzymic reduction at +50 mV vs Ag/AgCl. The cathodic current response could be measured in the presence of excess biotinylated glucose oxidase by incorporation of catalase in homogeneous solution to scavenge H2O2 generated in the bulk before it diffused to the electrode surface. The assay showed greatest sensitivity over the range of biotin concentrations 0.07 to 2 micrograms ml-1 in the presence of 10 micrograms ml-1 excess biotinylated glucose oxidase in the bulk solution.

Biosensing Techniques↗
PubMed1995

Amperometric detection of alkaline phosphatase activity at a horseradish peroxidase enzyme electrode based on activated carbon: potential application to electrochemical immunoassay.

Amperometric detection of alkaline phosphatase activity has been achieved using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the enzyme substrate. The production of hydrogen peroxide from the dephosphorylation of BCIP was measured using an activated carbon electrode with horseradish peroxidase immobilised to its surface by simple passive adsorption. This method was easily capable of measuring 10(-12) M alkaline phosphatase and had a calculated detection limit of 2.2 x 10(-14) M. The horseradish peroxidase electrode system was investigated further as a method for non-competitive electrochemical enzyme immunoassay using thyrotropin (TSH) as the model analyte. This was realised by co-immobilization to the electrode surface of both horseradish peroxidase and an anti-thyrotropin monoclonal antibody. After addition of the analyte, a second biotinylated anti-thyrotropin monoclonal antibody and the substrate, streptavidin-labelled alkaline phosphatase was added and the current (generated by enzyme channelling of hydrogen peroxide) measured as a function of TSH concentration. Thus, the activated carbon electrode was used as a combined immunological capture phase and amperometric detection system.

Alkaline Phosphatase↗
PubMed1995

Effect of calcitonin, elevated calcium and extracellular matrices on superoxide anion production by rat osteoclasts.

The electrochemical determination of superoxide anion generation by isolated mammalian osteoclasts was carried out by employing cytochrome c, immobilized at a surface-modified gold electrode. Osteoclasts cultured on bone generated 15-fold more superoxide anions than cells plated on plastic. Superoxide anion production by osteoclasts cultured on bone was almost completely abolished by salmon calcitonin, and partially abolished following exposure to elevated extracellular calcium. We therefore conclude that osteoclast activity is influenced substantially by the extracellular matrix.

Animals↗
PubMed1994

Amplified electrochemical immunoassay for thyrotropin using thermophilic beta-NADH oxidase.

The use of the highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase) from the thermophilic organism Thermus aquaticus in combination with alcohol dehydrogenase in an amperometric amplified immunoassay for thyrotropin (TSH) is described. NADH oxidase catalyses the oxidation of reduced nicotinamide adenine dinucleotide (NADH) with concomitant two electron reduction of di-oxygen to hydrogen peroxide. Hydrogen peroxide can be detected by oxidation at a platinum electrode poised at +650mV vs. Ag/AgCl. The enzyme amplification system described has advantages over existing amplification techniques in terms of sensitivity, specificity and operational pH dependence. The electrochemical enzyme-amplified assay for TSH was compared with a spectrophotometric enzyme-amplified system and with a non-amplified electrochemical immunoenzymometric TSH assay. The dynamic range of the electrochemical enzyme-amplified TSH immunoassay was 0.2-100 mIU/l, which was four times that of the enzyme-amplified spectrophotometric assay while the detection limits of both techniques were comparable.

Alkaline Phosphatase↗
PubMed1994

Potential glucose sensor for perioperative blood glucose control in diabetes mellitus.

A glucose sensor intended for future use in diabetic patients undergoing major surgery is described. It involves glucose oxidase immobilised at a platinised activated carbon electrode (PACE). The sensor gave a nonoxygen dependent amperometric response in whole undiluted blood and did not require the use of electron mediators. In vitro studies in protein containing buffer using a flow cell indicated current densities of approximately 160 nA mm-2 mM-1 and a linear response over the range 0-20 mM. The operational stability of the sensor was at least 49 h in continuous use. In addition the sensor had a 90-99% response time of 1 min when used at a flow rate of 3 ml min-1 and showed a temperature dependence of 2.4% C-1. The results reported suggest significant advantages of this approach, for future use as a perioperative intra-vascular sensor for diabetic subjects, over previously reported glucose sensors.

Biosensing Techniques↗
PubMed1993

Homogeneous amperometric immunoassay for theophylline in whole blood.

A homogeneous spectrophotometric EMIT immunoassay kit for the quantitation of theophylline in serum or plasma has been modified to produce a rapid, amperometric immunoassay requiring a 50 microliters whole blood sample. The basis of the detection system for the assay is the electrochemical oxidation of NADH produced by G6PDH-labelled theophylline at a potential of +150 mV vs Ag/AgCl using platinised activated carbon (PACE) electrodes. Comparison of the amperometric whole blood method with the conventional spectrophotometric plasma assay produced a reasonable correlation: y = 0.90x - 1.01, (r = 0.98, n = 12). The advantage of the new method is that simple and robust instrumentation can rapidly determine theophylline in whole blood with no sample pre-treatment or separation steps.

Biosensing Techniques↗
PubMed1993

Avidin-biotin based electrochemical immunoassay for thyrotropin.

A two site electrochemical enzyme immunoassay for thyrotropin (TSH) has been developed. This assay is based on the use of an immobilized capture antibody and a biotinylated second antibody. Detection is achieved via avidin labelled with alkaline phosphatase. The substrate 1-naphthyl phosphate was used and the product 1-naphthol was detected at disposable screen-printed carbon 8-electrode combs using specially designed instrumentation.

Alkaline Phosphatase↗
PubMed1992

Amperometric enzyme electrode for determination of theophylline in serum.

This paper describes an amperometric enzyme electrode for the rapid determination of theophylline in serum. The method is based on the catalysed oxidation of theophylline by the haem-containing enzyme theophylline oxidase. Results are presented for two approaches. First, ferrocene monocarboxylic acid was used as a mediator. The second-order rate constant was 1.1 x 10(3) 1 mol-1 s-1. Secondly, the organic conducting salt NMP.TCNQ was used to construct enzyme electrodes. These electrodes were employed for the rapid (60 s) measurement of theophylline in serum at a working potential of +100 mV versus Ag/AgCl. Linear calibration curves were obtained over the clinically relevant range (y = 0.13x + 0.22, n = 8). Caffeine, theobromine and 3-methylxanthine at levels up to 100 mg l-1 do not interfere and 1-methylxanthine shows cross-reactivity at concentrations greater than 50 mg l-1.

Biosensing Techniques↗
PubMed1992

Electrochemical sensors for direct reagentless measurement of superoxide production by human neutrophils.

Electrochemical sensors based on immobilised cytochrome c or superoxide dismutase for the measurement of superoxide radical production by stimulated neutrophils are described. Cytochrome c was immobilised covalently at a surface-modified gold electrode and by passive adsorption to novel platinised activated carbon electrodes (PACE). The reoxidation of cytochrome c at the electrode surface upon reduction by superoxide was monitored using both xanthine/xanthine oxidase and stimulated neutrophils as sources of the free radical. In addition, bovine Cu/Zn superoxide dismutase was immobilised to PACE by passive adsorption and superoxide, generated by xanthine/xanthine oxidase, detected by oxidation of hydrogen peroxide produced by the enzymic dismutation of the superoxide radical. A biopsy needle probe electrode based on cytochrome c immobilised at PACE and suitable for continuous monitoring of free radical production was constructed and characterised.

Biopsy↗
PubMed1991

Preparation and kinetic studies of immobilised yeast cytochrome c peroxidase.

Yeast cytochrome c peroxidase (CcP) was purified from baker's yeast and immobilised onto a nylon membrane. The kinetics of the soluble and immobilised forms of the enzyme were investigated for the catalysed oxidation of potassium ferrocyanide in the presence of H2O2 and m-chloroperoxybenzoic acid. The pH dependence of the two forms of the enzyme differed. Although both the soluble and the immobilised enzymes showed optimal activity at pH 6.2, a different kinetic behaviour was demonstrated. Both forms of the enzyme showed similar activity toward H2O2, although when m-chloroperoxybenzoic acid was replaced as the electron acceptor, the immobilised form of the enzyme had a reduced turnover number and an increased Km. The activation energy of immobilised CcP was greater in the presence of both H2O2 [16.6 kJ mol-1] and m-chloroperoxybenzoic acid [37.9 kJ mol-1] than for soluble CcP [11.4 and 23.4 kJ mol-1, respectively]. The activities of both soluble and immobilised CcP were greatly reduced above 45 degrees C, although at higher temperatures the immobilised enzyme retained a relatively greater percentage of its maximum activity.

Catalysis↗
PubMed1989

Thermostable reduced nicotinamide adenine dinucleotide oxidase: application to amperometric enzyme assay.

The use in amperometric enzyme assays of a highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase), from the thermophilic organism Thermus aquaticus is described. The enzyme catalyses the oxidation of reduced nicotinamide adenine dinucleotide with concomitant two-electron reduction of dioxygen to hydrogen peroxide. In addition the enzyme used a substituted ferrocene as an alternative mediator of electron transfer. Hydrogen peroxide was detected at +650 mV vs Ag/AgCl at a platinum electrode. The current produced by oxidation of hydrogen peroxide was directly proportional to NADH concentration. The enzyme was used in solution to reoxidize enzymatically generated NADH and served as a basis for amperometric enzyme amplification systems for immunoassay as well as for the detection of substrate concentration for oxidoreductase enzymes. In the presence of alcohol dehydrogenase a rapid production of current occurred upon addition of ethanol over a clinically significant range. Thermus aquaticus NADH oxidase appears to be ideally suited for future exploitation in amperometric sensors for oxidoreductase substrates, offering a number of advantages over previously reported methods.

Electrochemistry↗
PubMed1989

Application of the electrochemistry of cytochrome c to the measurement of superoxide radical production.

An electrochemical sensor for the measurement of the superoxide anion produced as a result of the respiratory burst of neutrophils is described. The reduction of cytochrome c by the superoxide ion was monitored using a surface-modified gold electrode. The current produced was shown to be superoxide-specific and proportional to the number of neutrophils in both purified neutrophil preparations and plasma.

Cytochrome c Group↗
PubMed1986

Early and late changes in sulphydryl group and copper protein concentrations and activities during drug treatment with aurothiomalate and auranofin.

Superoxide dismutase activity (SOD), plasma and lysate thiol concentrations (PSH and LSH), and caeruloplasmin oxidase activity (CP) reflect the underlying reduction-oxidation imbalance associated with rheumatoid arthritis (RA), and are believed to be involved in the protection of the cell against free radical activity. The early and late changes in these parameters have been observed and compared with standard clinical and biochemical assessments of disease activity in 90 patients with active RA, randomly assigned to receive either sodium aurothiomalate, auranofin, or auranofin placebo. An index based on clinical criteria was used to identify patients as responders or non-responders after 24 weeks of therapy. In the first six weeks of treatment a change in SOD activity and LSH concentration in a direction away from controls was followed by a return towards control levels in responders only. This suggests that in RA evidence of clinical improvement induced by gold drugs is preceded by an initial biochemical response in an inflammatory direction. The extracellular parameters PSH and CP did not show the same early response, but PSH levels in responders showed a slower change towards normal values, though at no time were values obtained that might suggest a complete remission. Thus the intracellular parameters appear to reflect an early effect of the drugs on cells which may possibly be of use in predicting the outcome of therapy, whereas the extracellular parameters provide confirmatory evidence for an eventual improvement.

Arthritis, Rheumatoid↗