Antioxidant inhibition of oxygen radicals for measurement of total antioxidant capacity in biological samples

Few methods for assessing total antioxidant capacity (TAC) include both the percentage of inhibition and the length of inhibition in the measurement. Available methods require above ambient constant temperature incubation, reaction preheating, and/or separate assays for testing hydrophilic and hydrophobic samples. We describe a high-throughput method, antioxidant inhibition of oxygen radicals (AIOR), that overcomes these difficulties. AIOR uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule, uroporphyrin I, which is delayed by the presence of antioxidants. The area under the curve is measured by a fluorescence spectrophotometer in a 96-well microplate format, and TAC results are expressed as millimole/liter Trolox equivalents. AIOR is performed at ambient temperature and is applicable to samples in either aqueous or common organic solvents. The reaction between uroporphyrin I and the peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be of first-order kinetics with a mean rate constant (k) of 0.0254. Applications to measure antioxidant capacity are demonstrated on individual chemicals and biological samples. The method has good linearity, within- and between-assay precision, and recovery.     

The Role of H<Superscript>+</Superscript>/OH<Superscript>−</Superscript> Channels in the Salt Stress Response of <Emphasis Type="Italic">Chara australis</Emphasis>

We investigate the electrophysiological salt stress response of the salt-sensitive charophyte Chara australis as a function of time in saline artificial pond water (saline APW) containing 50 mM NaCl and 0.1 mM CaCl₂. The effects are due to an increase in Na⁺ concentration rather than an increase in Cl⁻ concentration or medium osmolarity. A previous paper (Shepherd et al. Plant Cell Environ 31:1575–1591, 2008) described the rise in the background conductance and inhibition of proton pumping in saline APW in the first 60 min. Here we investigate the shift of membrane potential difference (PD) to levels above −100 mV and the change of shape of the current–voltage (I/V) profiles to upwardly concave. Arguing from thermodynamics, the I/V characteristics can be modeled by channels that conduct H⁺ or OH⁻. OH⁻ was chosen, as H⁺ required an unrealistic increase in the number/permeability of the channels at higher pH levels. Prolonged exposure to saline APW stimulated opening of more OH⁻ channels. Recovery was still possible even at a PD near −50 mV, with partial return of proton pumping and a decrease in OH⁻ current following APW wash. Upon change of pH from 7 to 9, the response was consistent with previously observed I/V characteristics of OH⁻ channels. For a pH change to 6, the response was transient before channel closure but could still be modeled. The consequences of opening of H⁺ or OH⁻ channels while the cell is under salt stress are discussed.     

The transport systems of Ventricaria ventricosa: hypotonic and hypertonic turgor regulation

In Acheta domesticus, the Malpighian tubules (Mt) are composed of three morphologically distinct regions (proximal, mid and distal), each consisting of a single cell type. The bulk of the Mt is composed of the midtubule, which shows the greatest response to corticotropin releasing factor-related diuretic peptides (CRF-DP). We know from previous laboratory studies that the second messenger cAMP and its analog dibutyryl cAMP (db-cAMP) cause an approximate doubling in the secretion rate and that this is accompanied by notable ultrastructural changes in the midtubule, especially membrane reorganization in the basal area and extensive vesiculation of the cytoplasm. In this study, we examined the morphological changes in membranes both at the cell surface and internally. By enzymatically removing the basal lamina, we examined the increase in spacing between infolded membranes initiated by db-cAMP stimulation. To examine the intracellular membranes, we used a technique developed for use in invertebrate tissues. This allowed the removal of the cytoplasm for high resolution scanning electron microscopy (HR-SEM) while maintaining the integrity of the lipid constituents of the cell. By using HR-SEM and confocal laser scanning microscopy (CLSM), we gained a unique three-dimensional perspective of the complexity of the internal membrane system of the A. domesticus Mt in both the unstimulated and db-cAMP-stimulated states.     

The proton pump,high pH channels,and excitation: voltage clamp studies of intact and perfused cells ofNitellopsis obtusa

Summary The current-voltage (I/V) and conductance-voltage (G/ V) characteristics were recorded for intact and perfused (tonoplast-free) cells ofNitellopsis obtusa. In the pH₀ range 5 to 8, the I/V profile was sigmoidal and the G/V profile exhibited a maximum — these characteristics are attributed to the proton pump at the plasmalemma. The pH₀ dependence in this range was very similar to that found inChara corallina. At very alkaline pH₀ (11.0) the high conductance due to H⁺/OH^– channels was observed in intact cells but not in perfused cells. Young plants ofNitellopsis did not display bands of calcification, but did exhibit pH banding patterns in petri dishes. The pH bands were less than 5mm wide. The excitation transients in intact cells featured two peaks near the excitation threshold, but more peaks could be observed in the p.d. (potential difference) range –90 to –60 mV. The amplitude of the transients was strongly inhibited at pH₀ 11.0. In the perfused cells the currents lacked complete inhibition at some p.d. levels, but still exhibited one or two peaks. At high pH₀ this lack of inactivation was accentuated. The addition of 5 mM TEA to the outside medium abolished the excitation transients in perfused cells.Abbreviations EGTA ethylene glycol-bis (-amino-ethylether)-N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethane sulfonic acid) - TEA tetraethyl ammonium     

The Effect of an Extracellular Mucilage on the Response to Osmotic Shock in the Charophyte Alga Lamprothamnium papulosum

We have used current/voltage (I/V) analysis to investigate the role played by extracellular mucilage in the cellular response to osmotic shock in Lamprothamnium papulosum. Cells lacking extracellular mucilage originated in a brackish environment (1/3 seawater). These were compared, first with cells coated with thick (∼50 μm) extracellular mucilage, collected from a marine lake, and second, with equivalent mucilaginous marine cells, treated with heparinase enzyme to disrupt the mucilage layer. Histochemical stains Toluidine Blue and Alcian Blue at low pH identified the major component of the extracellular mucilage as sulfated polysaccharides. Treating mucilage with heparinase removed the capacity for staining with cationic dyes at low pH, although the mucilage was not removed, and remained as a substantial unstirred layer. Cells lacking mucilage responded to hypotonic shock with depolarization (by ∼95 mV), cessation of cyclosis, due to transient opening of Ca²⁺ channels, and opening of Ca²⁺-activated Cl⁻ channels and K⁺ channels. Cell conductance transiently increased tenfold, but after 60 min was restored to the conductance prior to hypotonic shock. Mucilaginous cells depolarized by a small amount (∼18 mV), but Ca²⁺ channels failed to open in large enough numbers for cyclosis to cease. Likewise most Ca²⁺-activated Cl⁻ channels failed to open and conductance increased only ∼1.2-fold above the prehypotonic level. After 60 min conductance was less than the conductance prior to hypotonic shock. Heparinased mucilaginous cells recovered several aspects of the hypotonic response in cells lacking mucilage. These cells depolarized (by ∼103 mV); cyclosis ceased, indicating that Ca²⁺ channels had opened, and conductance increased to ∼4 times the value prior to hypotonic shock, indicating that Ca²⁺-activated Cl⁻ channels opened. However, after 60 min, these cells had neither restored membrane potential (and remained at positive values), nor decreased their conductance. It was not possible to determine whether K⁺ channels had opened. The heparinased cells recovered the normal hypotonic response of mucilaginous cells when heparinase was washed out. Apical seawater cells, which lacked mucilage, were unaffected by heparinase treatment. The results demonstrate that the presence of extracellular sulfated polysaccharide mucilage impacts upon the electrophysiology of the response to osmotic shock in Lamprothamnium cells. The role of such sulfated mucilages in marine algae and animal cells is compared and discussed. Received: 24 March 1998/Revised: 28 April 1999     

A pragmatic cluster randomised controlled trial to evaluate the safety, clinical effectiveness, cost effectiveness and satisfaction with point of care testing in a general practice setting – rationale, design and baseline characteristics

The UK has a strong tradition of innovative evaluative health care research. There are, however, considerable forces impeding collaboration between clinicians, academics, patients and their advocates and industry. This paper argues that, if the UK is to regain a position at the forefront of clinical research into evaluation of care, some of these forces need to be overcome. Now, with explicit encouragement from funders within the UK's NHS, it is urgent that all parties discover better ways of working together so that more broad and meaningful research can be produced in a timely fashion.