Effect of blood flow occlusion on corticospinal excitability during sustained low-intensity isometric elbow flexion.

Blood flow occlusion (BFO) has been used to study the influence of group III/IV muscle afferents after fatiguing exercise, but it is unknown how BFO-induced activity of these afferents affects motor cortical and motoneuronal excitability during low-intensity exercise. Therefore, the purpose of this study was to assess the acute effect of BFO on peripheral [maximal M wave (Mmax)], spinal [cervicomedullary motor evoked potential (CMEP) normalized to Mmax], and motor cortical [motor evoked potential (MEP) normalized to CMEP] excitability. Nine healthy men completed a sustained isometric contraction of the elbow flexors at 20% of maximal force under three conditions: 1) contractile failure with BFO, 2) a time-matched trial without restriction [free flow (FFiso)], and 3) contractile failure with free flow (FFfail). Time to failure for BFO (and FFiso) were ~80% shorter than that for FFfail (P < 0.05). For FFfail and FFiso, Mmax area decreased ~17% and ~7%, respectively (P < 0.05), with no change during BFO. CMEP/Mmax area increased ~226% and ~80% during BFO and FFfail, respectively (P < 0.05), with no change during FFiso (P > 0.05). The increase in normalized CMEP area was greater for BFO and FFfail compared with FFiso and for BFO compared with FFfail. MEP/CMEP area was not different among the protocols (P > 0.05) and increased ~64% with time (P < 0.05). It is likely that group III/IV muscle afferent feedback to the spinal cord modulates the large increase in motoneuronal excitability for the BFO compared with FFfail and FFiso protocols.NEW & NOTEWORTHY We have observed how blood flow occlusion modulates motor cortical, spinal, and peripheral excitability during and immediately after a sustained low-intensity isometric elbow flexion contraction to failure. We conclude that blood flow occlusion causes a greater and more rapid increase in motoneuronal excitability.

Species-Specific Detection of C. difficile Using Targeted Antibody Design.

Clostridium difficile is a Gram-positive, spore-forming bacterium that continues to present a worldwide problem in healthcare settings. The bacterium causes disease, the symptoms of which include diarrhea, fever, nausea, abdominal pain and even death. Despite the prevalence of the disease, the diagnosis of C. difficile infection is still challenging, with a variety of methods available, each varying in their effectiveness. In this work we sought to identify a new biomarker for C. difficile, develop affinity reagents and design a diagnostic assay for C. difficile infection which could be used in a typical two-step testing algorithm. Initially a bioinformatics pipeline was developed that identified a surface associated biomarker "AKDGSTKEDQLVDALA" present in all C. difficile strains sequenced to-date and unique to the C. difficile species. Monoclonal antibodies were subsequently raised against peptides corresponding to the biomarker sequence. During characterization studies, monoclonal antibody 521 (mAb521) was shown to bind all known C. difficile surface layer types, but not closely related strains. Surface plasmon resonance measurements were used to calculate an apparent equilibrium dissociation constant of 36.5 nM between the purified protein target containing the biomarker (surface layer protein A) and mAb521. We demonstrate a limit of detection of 12.4 ng/mL against surface layer protein A and 1.7 × 106 cells/mL in minimally processed C. difficile cultures. The utility of this computational approach to antibody design for diagnostic tests is the ability to produce antibodies that can act as universal species identifiers while mitigating the likelihood of false-positive detection by intelligently screening potential biomarkers against RefSeq data for other nontarget bacteria.

Whole-body and splanchnic amino acid metabolism in sheep during an acute endotoxin challenge.

Supplemented protein or specific amino acids (AA) are proposed to help animals combat infection and inflammation. The current study investigates whole-body and splanchnic tissue metabolism in response to a lipopolysaccharide (LPS) challenge with or without a supplement of six AA (cysteine, glutamine, methionine, proline, serine and threonine). Eight sheep were surgically prepared with vascular catheters across the gut and liver. On two occasions, four sheep were infused through the jugular vein for 20 h with either saline or LPS from Escherichia coli (2 ng/kg body weight per min) in a random order, plus saline infused into the mesenteric vein; the other four sheep were treated with saline or LPS plus saline or six AA infused via the jugular vein into the mesenteric vein. Whole-body AA irreversible loss rate (ILR) and tissue protein metabolism were monitored by infusion of [ring-2H2]phenylalanine. LPS increased (P<0·001) ILR (+17 %), total plasma protein synthesis (+14 %) and lymphocyte protein synthesis (+386 %) but decreased albumin synthesis (-53 %, P=0·001), with no effect of AA infusion. Absorption of dietary AA was not reduced by LPS, except for glutamine. LPS increased the hepatic removal of leucine, lysine, glutamine and proline. Absolute hepatic extraction of supplemented AA increased, but, except for glutamine, this was less than the amount infused. This increased net appearance across the splanchnic bed restored arterial concentrations of five AA to, or above, values for the saline-infused period. Infusion of key AA does not appear to alter the acute period of endotoxaemic response, but it may have benefits for the chronic or recovery phases.

Outcomes of patients with non-melanoma solid tumours receiving self-funded pembrolizumab at Chris O'Brien Lifehouse.

BACKGROUND: Immunotherapy agents show anti-cancer activity in several solid cancers. Efficacy in non-melanoma solid tumours for non-approved indications is unknown. AIM: To evaluate patient and disease characteristics, rate and duration of response, and toxicity of self-funded pembrolizumab in patients with non-melanoma solid cancers. METHOD: Retrospective review describing outcomes and toxicity of self-funded pembrolizumab in patients with non-melanoma solid cancers treated at Chris O'Brien Lifehouse. RESULTS: From April 2015 to December 2015, 21 patients received or were planned to receive self-funded pembrolizumab. The median age was 50 years (16-76), 28 and 10% had an Eastern Cooperative Oncology Group performance status of 2, and 3-4 respectively. Sixty-two percent received at least two to four lines of prior drug treatment. Median follow-up was 3.0 months (range, 0.4-9.6). Fourteen (67%) patients requested pembrolizumab. Pembrolizumab was clinician offered for 7 (33%) patients. Patients who requested pembrolizumab had worse outcomes. Three patients died before receiving pembrolizumab. Of the 18 patients that received at least one dose, a partial response was observed in 3 (17%). Progressive disease occurred in 83%. Four patients received only one cycle of pembrolizumab and died after a median of 27 days (range 13-43). Immune-related adverse events of any grade occurred in 33%. No grade 3-4 events were observed. CONCLUSION: Pembrolizumab was well tolerated. Meaningful responses were observed in 17% of treated patients. Response continues after 5-6.5 months follow-up in 11% and >8 months of follow-up for the other responding patient. Financial impact to the patient can be substantial. Outcomes for 33% were poor with three patients dying prior to receiving therapy and four dying within weeks of receiving one dose. This highlights issues regarding the careful selection of patients, futility of anti-cancer therapy at the end-of-life and patients' perceived benefit of receiving this therapy.

Examining non-syndromic autosomal recessive intellectual disability (NS-ARID) genes for an enriched association with intelligence differences.

Two themes are emerging regarding the molecular genetic aetiology of intelligence. The first is that intelligence is influenced by many variants and those that are tagged by common single nucleotide polymorphisms account for around 30% of the phenotypic variation. The second, in line with other polygenic traits such as height and schizophrenia, is that these variants are not randomly distributed across the genome but cluster in genes that work together. Less clear is whether the very low range of cognitive ability (intellectual disability) is simply one end of the normal distribution describing individual differences in cognitive ability across a population. Here, we examined 40 genes with a known association with non-syndromic autosomal recessive intellectual disability (NS-ARID) to determine if they are enriched for common variants associated with the normal range of intelligence differences. The current study used the 3511 individuals of the Cognitive Ageing Genetics in England and Scotland (CAGES) consortium. In addition, a text mining analysis was used to identify gene sets biologically related to the NS-ARID set. Gene-based tests indicated that genes implicated in NS-ARID were not significantly enriched for quantitative trait loci (QTL) associated with intelligence. These findings suggest that genes in which mutations can have a large and deleterious effect on intelligence are not associated with variation across the range of intelligence differences.

Risk factors for hospitalisation and associated costs among patients with hepatitis A associated with imported pomegranate arils, United States, 2013.

OBJECTIVES: To assess hospitalisation risk factors and economic effects associated with a multistate hepatitis A outbreak in 2013. STUDY DESIGN: Retrospective case series. METHODS: Eligible outbreak-related cases confirmed by September 1, 2013, were defined as acute hepatitis symptoms and positive IgM anti-hepatitis A during March 15-August 12 among patients who consumed the food vehicle or had the outbreak genotype. We reviewed medical records, comparing demographic and clinical characteristics among hospitalized and non-hospitalized patients; we used logistic regression analysis to identify factors associated with hospitalization. We interviewed patients regarding symptom duration and healthcare usage and estimated per-patient and total costs. Health departments reported outbreak-related personnel hours. RESULTS: Medical records were reviewed for 147/159 (92%) eligible patients; median age was 48 (range: 1-84) years, and 64 (44%) patients were hospitalized. Having any chronic medical condition was independently associated with hospitalisation (odds ratio, 3.80; 95% confidence interval, 1.68-8.62). Interviews were completed for 114 (72%) eligible patients; estimated per-patient cost of healthcare and productivity loss was $13,467 for hospitalized and $2138 for non-hospitalized patients and $1,304,648 for all 165 outbreak-related cases. State and local public health personnel expenditures included 82 h and $3221/outbreak-related case. CONCLUSIONS: Hospitalisations in this outbreak were associated with chronic medical conditions and resulted in substantial healthcare usage and lost productivity. These data can be used to inform future evaluation of expansion of hepatitis A vaccination recommendations to include adults with chronic medical conditions.

Progression of KRAS mutant pancreatic adenocarcinoma during vemurafenib treatment in a patient with metastatic melanoma.

Vemurafenib is a tyrosine kinase inhibitor of BRAF that prolongs survival in patients with BRAF V600-mutant metastatic melanoma. Secondary cutaneous malignancies are a well-documented toxicity of vemurafenib, thought to be mediated by enhanced ERK signalling in BRAF wild-type, RAS-mutant cells. Vemurafenib could also promote growth of non-cutaneous secondary malignancies by a similar mechanism. We present a case of an individual who received vemurafenib for metastatic melanoma and experienced rapid growth of a pre-existing KRAS-mutant pancreatic adenocarcinoma.

Twitch interpolation: superimposed twitches decline progressively during a tetanic contraction of human adductor pollicis.

The assessment of voluntary activation of human muscles usually depends on measurement of the size of the twitch produced by an interpolated nerve or cortical stimulus. In many forms of fatiguing exercise the superimposed twitch increases and thus voluntary activation appears to decline. This is termed 'central' fatigue. Recent studies on isolated mouse muscle suggest that a peripheral mechanism related to intracellular calcium sensitivity increases interpolated twitches. To test whether this problem developed with human voluntary contractions we delivered maximal tetanic stimulation to the ulnar nerve (≥60 s at physiological motoneuronal frequencies, 30 and 15 Hz). During the tetani (at 30 Hz) in which the force declined by 42%, the absolute size of the twitches evoked by interpolated stimuli (delivered regularly or only in the last second of the tetanus) diminished progressively to less than 1%. With stimulation at 30 Hz, there was also a marked reduction in size and area of the interpolated compound muscle action potential (M wave). With a 15 Hz tetanus, a progressive decline in the interpolated twitch force also occurred (to ∼10%) but did so before the area of the interpolated M wave diminished. These results indicate that the increase in interpolated twitch size predicted from the mouse studies does not occur. Diminution in superimposed twitches occurred whether or not the M wave indicated marked impairment at sarcolemmal/t-tubular levels. Consequently, the increase in superimposed twitch, which is used to denote central fatigue in human fatiguing exercise, is likely to reflect low volitional drive to high-threshold motor units, which stop firing or are discharging at low frequencies.

Prediction of outcome of early ER+ breast cancer is improved using a biomarker panel, which includes Ki-67 and p53.

BACKGROUND: The aim of this study is to determine whether immunohistochemical (IHC) assessment of Ki67 and p53 improves prognostication of oestrogen receptor-positive (ER+) breast cancer after breast-conserving therapy (BCT). In all, 498 patients with invasive breast cancer from a randomised trial of BCT with or without tumour bed radiation boost were assessed using IHC. METHODS: The ER+ tumours were classified as 'luminal A' (LA): ER+ and/or PR+, Ki-67 low, p53-, HER2- or 'luminal B' (LB): ER+ and/or PR+and/or Ki-67 high and/or p53+ and/or HER2+. Kaplan-Meier and Cox proportional hazards methodology were used to ascertain relationships to ispilateral breast tumour recurrence (IBTR), locoregional recurrence (LRR), distant metastasis-free survival (DMFS) and breast cancer-specific survival (BCSS). RESULTS: In all, 73 patients previously LA were re-classified as LB: a greater than four-fold increase (4.6-19.3%) compared with ER, PR, HER2 alone. In multivariate analysis, the LB signature independently predicted LRR (hazard ratio (HR) 3.612, 95% CI 1.555-8.340, P=0.003), DMFS (HR 3.023, 95% CI 1.501-6.087, P=0.002) and BCSS (HR 3.617, 95% CI 1.629-8.031, P=0.002) but not IBTR. CONCLUSION: The prognostic evaluation of ER+ breast cancer is improved using a marker panel, which includes Ki-67 and p53. This may help better define a group of poor prognosis ER+ patients with a greater probability of failure with endocrine therapy.

BAG-1 predicts patient outcome and tamoxifen responsiveness in ER-positive invasive ductal carcinoma of the breast.

BAG-1 (bcl-2-associated athanogene) enhances oestrogen receptor (ER) function and may influence outcome and response to endocrine therapy in breast cancer. We determined relationships between BAG-1 expression, molecular phenotype, response to tamoxifen therapy and outcome in a cohort of breast cancer patients and its influence on tamoxifen sensitivity in MCF-7 breast cancer cells in vitro. Publically available gene expression data sets were analysed to identify relationships between BAG-1 mRNA expression and patient outcome. BAG-1 protein expression was assessed using immunohistochemistry in 292 patients with invasive ductal carcinoma and correlated with clinicopathological variables, therapeutic response and disease outcome. BAG-1-overexpressing MCF-7 cells were treated with antioestrogens to assess its effects on cell proliferation. Gene expression data demonstrated a consistent association between high BAG-1 mRNA and improved survival. In ER+ cancer (n=189), a high nuclear BAG-1 expression independently predicted improved outcome for local recurrence (P=0.0464), distant metastases (P=0.0435), death from breast cancer (P=0.009, hazards ratio 0.29, 95% CI: 0.114-0.735) and improved outcome in tamoxifen-treated patients (n=107; P=0.0191). BAG-1 overexpression in MCF-7 cells augmented antioestrogen-induced growth arrest. A high BAG-1 expression predicts improved patient outcome in ER+ breast carcinoma. This may reflect both a better definition of the hormone-responsive phenotype and a concurrent increased sensitivity to tamoxifen.

The key hypoxia regulated gene CAIX is upregulated in basal-like breast tumours and is associated with resistance to chemotherapy.

Basal-like tumours account for 15% of invasive breast carcinomas and are associated with a poorer prognosis and resistance to therapy. We hypothesised that this aggressive phenotype is because of an intrinsically elevated hypoxic response. Microarrayed tumours from 188 patients were stained for hypoxia-inducible factor (HIF)-1alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and factor inhibiting HIF (FIH)-1, and carbonic anhydrase (CA) IX stained in 456 breast tumours. Tumour subtypes were correlated with standard clincopathological parameters as well as hypoxic markers. Out of 456 tumours 62 (14%) tumours were basal-like. These tumours were positively correlated with high tumour grade (P<0.001) and were associated with a significantly worse disease-free survival compared with luminal tumours (P<0.001). Fifty percent of basal-like tumours expressed HIF-1alpha, and more than half expressed at least one of the PHD enzymes and FIH-1. Basal-like tumours were nine times more likely to be associated with CAIX expression (P<0.001) in a multivariate analysis. Carbonic anhydrase IX expression was positively correlated with tumour size (P=0.005), tumour grade (P<0.001) and oestrogen receptor (ER) negativity (P<0.001). Patients with any CAIX-positive breast tumour phenotype and in the basal tumour group had a significantly worse prognosis than CAIX-negative tumours when treated with chemotherapy (P<0.001 and P=0.03, respectively). The association between basal phenotype and CAIX suggests that the more aggressive behaviour of these tumours is partly due to an enhanced hypoxic response. Further, the association with chemoresistance in CAIX-positive breast tumours and basal-like tumours in particular raises the possibility that targeted therapy against HIF pathway or downstream genes such as CAs may be an approach to investigate for these patients.

Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids.

Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein-phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter.

Altered muscle development and expression of the insulin-like growth factor system in growth retarded fetal pigs.

We have used a porcine model of spontaneous differential fetal growth to investigate the effects of fetal size on muscle development. We hypothesized that altered muscle development may occur in small fetuses as a consequence of modified expression of selected genes of the insulin-like growth factor system. We examined the development of the Longissimus muscle (m. Longissimus) in small fetuses and their average sized littermates. We collected small for gestational age fetuses and their average sized sibling on days 45, 65 and 100 of gestation (term is 113-116 days). Small fetuses had significantly lower body weight at all three stages of gestation (p<0.05) and significantly reduced secondary to primary muscle fibre ratio in m. Longissimus on day 100 (p<0.05) compared to their littermates. On day 65, the expression of insulin-like growth factor receptor 1 and insulin-like growth factor binding protein 3 were significantly higher (p<0.05) in m. Longissimus of the small fetuses compared with their average sized littermates. On day 100, the expression of insulin-like growth factor receptor 1 remained significantly higher (p=0.001), in addition to significantly higher levels of insulin-like growth factor receptor 2 and insulin-like growth factor binding protein 5 in the small fetuses (p<0.05). No difference in levels of myogenin was observed between the small and average sized littermates. In conclusion, we demonstrate that reduced fetal muscle development is associated with an increased expression of several genes of the insulin-like growth factor system in small fetuses in mid to late gestation.

Activation of peroxisome proliferator-activated receptor gamma and retinoid X receptor results in net depletion of cellular cholesteryl esters in macrophages exposed to oxidized lipoproteins.

OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor, has pleiotropic effects, including regulation of macrophage differentiation and lipid homeostasis. The PPARgamma ligands, thiazolidinediones (TZDs), attenuate atherosclerosis in mice by uncertain mechanisms. The objective of this study was to determine whether activation of PPARgamma or its obligate heterodimer, retinoid X receptor (RXR), modulates macrophage foam cell formation induced by oxidized (ox) lipoproteins. METHODS AND RESULTS: Incubation of THP-1 macrophages with oxHTG-VLDL, oxREM, or oxLDL increased cellular cholesteryl ester over 6-fold. Preincubation with the TZD, ciglitazone, the RXR-specific ligand, 9-cis retinoic acid (9cRA) or the combination reduced CE mass accumulation by up to 65%. Ciglitazone and 9cRA increased CD36 mRNA (up to 4-fold); however, uptake of [125I]oxLDL was only modestly enhanced (up to 1.8-fold) becaues of a concomitant PPARgamma:RXR-induced decrease in SRAI/II activity (up to 40%). This suggested that PPARgamma:RXR activation inhibited cholesteryl ester accumulation by enhancing cholesterol efflux. Ciglitazone and 9cRA were found to increase the expression of ATP-binding cassette proteins A1 and G1, resulting in enhanced cholesterol efflux to lipoprotein-deficient serum, apoAI and HDL3. CONCLUSIONS: PPARgamma and/or RXR activation inhibit foam cell formation through enhanced cholesterol efflux despite increased oxLDL uptake. These observations explain the reduced atherosclerosis in TZD-treated mice and may extend the therapeutic implications of these ligands.

alpha-melanocyte-stimulating hormone modulates nitric oxide production in melanocytes.

We have previously observed that melanocytes produce nitric oxide in response to ultraviolet radiation and lipopolysaccharide and in this study have examined how these responses are affected by alpha-melanocyte-stimulating hormone. Nitric oxide production by cultured cells was measured electrochemically in real time using an ISO-nitric oxide sensor probe. B16 mouse melanoma cells released nitric oxide in response to lipopolysaccharide and the effects were enhanced in cells that had been grown in the presence of 10-11-10-9 M alpha-melanocyte-stimulating hormone prior to stimulation. At concentrations in excess of 10-9 M alpha-melanocyte-stimulating hormone decreased nitric oxide production. Preincubation with lipopolysaccharide, a well-known inducer of inducible nitric oxide synthase, also increased nitric oxide production but this response was reduced by alpha-melanocyte-stimulating hormone. alpha-Melanocyte-stimulating hormone also increased the levels of nitric oxide produced in response to ultraviolet radiation (20-100 mJ per cm2) in B16 cells. The same effect was seen in human melanocytes and as this was inhibited by aminoguanidine would appear to involve an induction of inducible nitric oxide synthase. Reverse transcription-polymerase chain reaction showed that melanocytic cells express inducible nitric oxide synthase mRNA. Western blotting analysis and immunocytochemistry confirmed the presence of inducible nitric oxide synthase protein in B16 cells and FM55 human melanoma cells and that the levels were increased in response to alpha-melanocyte-stimulating hormone. alpha-Melanocyte-stimulating hormone, however, decreased inducible nitric oxide synthase protein expression, which occurred in response to lipopolysaccharide. These results suggest that alpha-melanocyte-stimulating hormone regulates nitric oxide production in melanocytic cells by modulating the induction of inducible nitric oxide synthase. Additional experiments showed that nitric oxide increased melanin production by B16 cells and human melanocytes. This is in keeping with a melanogenic role for nitric oxide but whether its production by melanocytes in response to alpha-melanocyte-stimulating hormone is associated with such a role or whether it has some other significance relating to melanocyte differentiation or in mediating immunomodulatory actions of alpha-melanocyte-stimulating hormone remains to be seen.

Superoxide generation from constitutive nitric oxide synthase in astrocytes in vitro regulates extracellular nitric oxide availability.

Oxygen-derived free radicals play an important role in the physiology and pathophysiology of brain cell function. Because of their labile nature, however, it has been difficult to investigate their actions directly. This problem has been addressed, in primary rat brain cell cultures, in this study by utilization of two novel electrochemical sensors. It has been demonstrated that extracellular superoxide originates from the astrocytic subpopulation in a calcium/calmodulin dependent manner and responds to constitutive nitric oxide synthase inhibition. The results indicate a novel function for the astrocytic constitutive nitric oxide synthase in regulating extracellular superoxide release and, therefore, controlling neuronal nitric oxide availability.

Direct, real-time sensing of free radical production by activated human glioblastoma cells.

Primary brain injury initiates a cascade of events which result in secondary brain damage. Although, at present, the biochemical and molecular mechanisms of nerve cell death are not well understood, sufficient evidence now exists to implicate free radicals in this brain injury response. In the light of the current understanding on the role of free radicals in cell mortality, we report on the use of two specific sensors, which we use to measure the direct, simultaneous and real time electrochemical detection of both superoxide (O2.-) and nitric oxide (NO), produced by activated glioblastoma cells. The development and application of these novel methods has enabled us to show that both the cytokine-mediated induction of the enzymes responsible for the generation of these radical species, and the metabolic requirements of the cell can modulate cell messenger release. Importantly, the data collected provides dynamic information on the time course of free radical production, as well as their interactions and their involvement in the process of cell death. In particular, one of the major advances afforded by this technology is the demonstration that suppression of one of either of the two cellular generated radical species (NO and O2.-) leads directly to a corresponding increase in the species that was not being deliberately inhibited or scavenged. This finding may indicate a mechanism involving inter-enzyme regulation of free radical production in glial cells (a phenomenon which may, in future, also be shown to operate in other relevant cell models).

Superoxide electrode based on covalently immobilized cytochrome c: modelling studies.

We have recently described an optimised electrode for the detection of enzymatic and cellular superoxide (O2*-) production based on cytochrome c immobilized covalently at a surface-modified gold electrode and applied this system to the study of free radical production by activated human glioblastoma cells. In this paper we report the development of a mathematical model for the O2*- electrode responding to enzymically produced O2*- which should enable the determination of absolute concentrations of O2*- in biological systems when this electrode is employed for direct, real-time monitoring of free radical release and interactions.

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.