Echinacea intake induces an immune response through altered expression of leucocyte hsp70, increased white cell counts and improved erythrocyte antioxidant defences

Objective: To study the effect of Echinacea tablets on the expression of leucocyte heat shock protein 70 (hsp70), erythrocyte haemolysis, plasma antioxidant status, serum chemistry, haematological values and plasma alkylamide concentrations. Method: Eleven healthy individuals (26-61 years of age) were evaluated at baseline (day 1) and on day 15 after consuming two commercially blended Echinacea tablets daily for 14 days. Results: Echinacea supplementation enhanced the fold increase in leucocyte hsp70 expression after a mild heat shock (P=0.029). White cell counts (WCC) were also increased (P=0.043). We also observed a preventative effect against free radical induced erythrocyte haemolysis (P=0.006) indicative of an antioxidant effect. Conclusion: The pilot study suggests that Echinacea may invoke an immune response through altered expression of hsp70 and increased WCC.

Study of the calcification of PHEMA hydrogels using a two compartment permeation cell

To simulate the process of calcification in hydrogel implants, particularly calcification inside hydrogels, in vitro experiments using two compartment permeation cells have been performed. PHEMA hydrogel membranes were synthesized by free radical polymerization in bulk. The permeability and diffusion coefficient for Ca2+ ions at 37 ° C were determined using Fick's laws of diffusion. It was evident that Ca2+ ions either from CaCl2 or SBF solutions may diffuse through PHEMA hydrogel membranes. The fort-nation of calcium phosphate deposits inside the hydrogel was observed and attributed to a heterogeneous nucleation from diffusing calcium and phosphate ions. The morphology of the deposits both on the surface and inside the hydrogels was found to be similar, i.e. spherical aggregates with a diameter of less than one micron. © 2005 Elsevier B.V. All rights reserved.

Physiological role of carnosine in contracting muscle

High-intensity exercise leads to reductions in muscle substrates (ATP, PCr, and glycogen) and a subsequent accumulation of metabolites (ADP, Pi, H+, and M2+) with a possible increase in free radical production. These factors independently and collectively have deleterious effects on muscle, with significant repercussions on high-intensity performance or training sessions. The effect of carnosine on overcoming muscle fatigue appears to be related to its ability to buffer the increased H+ concentration following high-intensity work. Carnosine, however, has other roles such as an antioxidant, a metal chelator, a Ca2+ and enzyme regulator, an inhibitor of protein glycosylation and protein-protein cross-linking. To date, only 1 study has investigated the effects of carnosine supplementation (not in pure form) on exercise performance in human subjects and found no improvement in repetitive high-intensity work. Much data has come from in vitro work on animal skeletal muscle fibers or other components of muscle contractile mechanisms. Thus further research needs to be carried out on humans to provide additional understanding on the effects of carnosine in vivo.

Antioxidant requirements of endurance athletes: Implications for health

This review discusses studies investigating the effects of antioxidant supplementation on exercise-induced oxidative stress with a focus on the health implications. The aim is to determine antioxidant requirements for endurance athletes. Overall, differences in methodology make it difficult to compare the relatively small number of published studies on this topic. The types of studies needed to more adequately assess the health effects of antioxidant supplements in athletes (long-term interventions with hard end points) have not been done. Therefore, there is currently insufficient evidence to recommend antioxidant supplements for endurance athletes.

Diel 'tuning' of coral metabolism: physiological responses to light cues

Hermatypic-zooxanthellate corals track the diel patterns of the main environmental parameters temperature, UV and visible light - by acclimation processes that include biochemical responses. The diel course of solar radiation is followed by photosynthesis rates and thereby elicits simultaneous changes in tissue oxygen tension due to the shift in photosynthesis/respiration balance. The recurrent patterns of sunlight are reflected in fluorescence yields, photosynthetic pigment content and activity of the two protective enzymes superoxide dismutase (SOD) and catalase (CAT), enzymes that are among the universal defenses against free radical damage in living tissue. All of these were investigated in three scleractinian corals: Favia favus, Plerogyra sinuosa and Goniopora lobata. The activity of SOD and CAT in the animal host followed the course of solar radiation, increased with the rates of photosynthetic oxygen production and was correlated with a decrease in the maximum quantum yield of photochemistry in Photosystem H (PSII) (Delta F'/F-m'). SOD and CAT activity in the symbiotic algae also exhibited a light intensity correlated pattern, albeit a less pronounced one. The observed rise of the free-radical-scavenger enzymes, with a time scale of minutes to several hours, is an important protective mechanism for the existence and remarkable success of the unique cnidarian-dinoflagellate associations, in which photosynthetic oxygen production takes place within animal cells. This represents a facet of the precarious act of balancing the photosynthetic production of oxygen by the algal symbionts with their destructive action on all living cells, especially those of the animal host.

The impact of spectral composition and light periodicity on the activity of two antioxidant enzymes (SOD and CAT) in the coral Favia favus

In oligotrophic waters the light spectrum is mostly blue, and therefore the physiological and biochemical responses to blue light occurring in the coral tissue and in the symbiotic algae are important. Examination of the wavelength dependence of two free radical scavenger enzyme activity revealed an increase in activity in the blue light range (440-480 nm) compared to the red (640680 nm) in the full visible light (400-700 nm) range. These data show for the first time the relationship between the action spectra of photosynthesis and the activity of two main antioxidant enzymes in the symbiotic coral Favia favus. It was found that in the animal (host) the enzyme response to the spectral distribution of light was higher than that of the zooxanthellae, probably due to accumulation of free radicals within the host tissue. Furthermore, we found that the activity of these enzymes is affected in nature by the length of the day and night, and in the laboratory, by the duration of the illumination. Changes in the pigment concentrations were also observed in response to growth under the blue region and the whole PAR spectrum, while fluorescence measurements with the fast repetition rate fluorometer (FRRF) showed a decrease in the sigma cross section and a decrease in the quantum yield also in the blue part of the spectrum. These changes of scavenger enzymes activity, pigment concentration and fluorescence yield at different light spectra are vital in acclimatization and survival of corals in shallow water environments with high light radiation. (c) 2005 Elsevier B.V. All rights reserved.

Ultra-endurance exercise and oxidative damage: Implications for cardiovascular health

At least 30 minutes of moderate-intensity physical activity accumulated on most, preferably all days is considered the minimum level necessary to reduce the risk of developing cardiovascular disease. Despite an unclear explanation, some epidemiological data paradoxically suggest that a very high volume of exercise is associated with a decrease in cardiovascular health. Although ultra-endurance exercise training has been shown to increase antioxidant defences (and therefore confer a protective effect against oxidative stress), an increase in oxidative stress may contribute to the development of atherosclerosis via oxidative modification of low-density lipoprotein (LDL). Research has also shown that ultra-endurance exercise is associated with acute cardiac dysfunction and injury, and these may also be related to an increase in free radical production. Longitudinal studies are needed to assess whether antioxidant defences are adequate to prevent LDL oxidation that may occur as a result of increased free radical production during very high volumes of exercise. In addition, this work will assist in understanding the accrued effect of repeated ultra-endurance exercise-induced myocardial damage.

Mechanism and kinetics of dithiobenzoate-mediated RAFT polymerization. I. The current situation

Investigations into the kinetics and mechanism of dithiobenzoate-mediated Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerizations, which exhibit nonideal kinetic behavior, such as induction periods and rate retardation, are comprehensively reviewed. The appreciable uncertainty in the rate coefficients associated with the RAFT equilibrium is discussed and methods for obtaining RAFT-specific rate coefficients are detailed. In addition, mechanistic studies are presented, which target the elucidation of the fundamental cause of rate retarding effects. The experimental and theoretical data existing in the literature are critically evaluated and apparent discrepancies between the results of different studies into the kinetics of RAFT polymerizations are discussed. Finally, recommendations for further work are given. (c) 2006 Wiley Periodicals, Inc.

Homocysteine from endothelial cells promotes LDL nitration and scavenger receptor uptake

We recently reported that methionine-loaded human umbilical vein endothelial cells (HUVECs) exported homocysteine (Hcy) and were associated with hydroxyl radical generation and oxidation of lipids in LDL. Herein we have analysed the Hcy-induced posttranslational modifications (PTMs) of LDL protein. PTMs have been characterised using electrophoretic mobility shift, protein carbonyl ELISA, HPLC with electrochemical detection and Western blotting of 3-nitrotyrosine, and LDL uptake by scavenger receptors on monocyte/macrophages. We have also analysed PTMs in LDL isolated from rheumatoid (RA) and osteo-(OA) arthritis patients with cardiovascular disease (CVD). While reagent Hcy (<50 μM) promoted copper-catalysed LDL protein oxidation, Hcy released from methionine-loaded HUVECs promoted LDL protein nitration. In addition, LDL nitration was associated with enhanced monocyte/macrophage uptake when compared with LDL oxidation. LDL protein nitration and uptake by monocytes, but not carbonyl formation, was elevated in both RA and OA patients with CVD compared with disease-matched patients that had no evidence of CVD. Moreover, a direct correlation between plasma total Hcy (tHcy) and LDL uptake was observed. The present studies suggest that elevated plasma tHcy may promote LDL nitration and increased scavenger receptor uptake, providing a molecular mechanism that may contribute to the clinical link between CVD and elevated plasma tHcy. © 2005 Elsevier Inc. All rights reserved.

Antioxidants and protein oxidation

Proteins are susceptible to oxidation by reactive oxygen species, where the type of damage induced is characteristic of the denaturing species. The induction of protein carbonyls is a widely applied biomarker, arising from primary oxidative insult. However, when applied to complex biological and pathological conditions it can be subject to interference from lipid, carbohydrate and DNA oxidation products. More recently, interest has focused on the analysis of specific protein bound oxidised amino acids. Of the 22 amino acids, aromatic and sulphydryl containing residues have been regarded as being particularly susceptible to oxidative modification, with L-DOPA from tyrosine, ortho-tyrosine from phenylalanine; sulphoxides and disulphides from methionine and cysteine respectively; and kynurenines from tryptophan. Latterly, the identification of valine and leucine hydroxides, reduced from hydroperoxide intermediates, has been described and applied. In order to examine the nature of oxidative damage and protective efficacy of antioxidants the markers must be thoroughly evaluated for dosimetry in vitro following damage by specific radical species. Antioxidant protection against formation of the biomarker should be demonstrated in vitro. Quantification of biomarkers in proteins from normal subjects should be within the limits of detection of any analytical procedure. Further to this, the techniques for isolation and hydrolysis of specific proteins should demonstrate that in vitro oxidation is minimised. There is a need for the development of standards for quality assurance material to standardise procedures between laboratories. At present, antioxidant effects on protein oxidation in vivo are limited to animal studies, where dietary antioxidants have been reported to reduce dityrosine formation during rat exercise training. Two studies on humans have been reported last year. The further application of these methods to human studies is indicated, where the quality of the determinations will be enhanced through inter-laboratory validation.

Immunochemical detection of glyoxal DNA damage

The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.

Molecular and cellular effects of ultraviolet light-induced genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

Evidence for sensitisation of DNA to oxidative damage during isolation

The oxidative base lesion 8-oxo-deoxyguanosine (8-oxo-dG) has been identified in DNA isolated from normal tissue and may occur at elevated levels during disease. However, the use of phenol during DNA extraction may artificially elevate the detected levels of this lesion. Herein, we have performed a comparative methodological study using both pronase E and phenol extraction techniques; native or oxidatively stressed DNA was isolated to determine the validity of each extraction technique for the subsequent determination of 8-oxo-dG. Whilst the yields of DNA were comparable, after pronase E extraction there was no detectable induction of 8-oxo-dG in reextracted naked DNA or peripheral blood mononuclear cell DNA that had been oxidatively stressed. However, phenol extraction enhanced the basal levels of 8-oxo-dG detected, and also induced a significant increase in levels of the modified base after exposure to oxidative stress. The latter was dependent on the presence of foetal calf serum in the extracellular medium. We have confirmed that phenol extraction sensitises native DNA to subsequent oxidative damage. In addition, this work shows that the extent of sensitisation occurring during phenol extraction varies with the degree of oxidative damage already incurred and infers that labile guanine sites generated during oxidative stress may be detected as 8-oxo-dG residues after phenol extraction.

Studies on the neurotoxicity of 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) in SH-SY5Y cells

We have studied the hypothesis that 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) is neurotoxic. Salsolinol induced a significant time and dose related inhibition of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction, and increased lactate dehydrogenase release (LDH) release from human SH-SY5Y neuroblastoma cells, at concentrations within the range of 1-methyl-4-phenylpyridinium (MPP+) cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of antioxidants, monoamine oxidase inhibitors or imipramine. In confluent monolayers, salsolinol stimulated catecholamine uptake with EC50 values of 17 muM and 11 muM, for noradrenaline and dopamine, respectively. Conversely, at concentrations above 100 muM, salsolinol inhibited the uptake of noradrenaline and dopamine, with IC50 values of 411 muM and 379 muM, respectively. The inhibition of catecholamine uptake corresponded to the increase displacement of [3H]nisoxetine from the uptake 1 site by salsolinol, as the Ki (353 muM) for displacement was similar to the IC50 (411 and 379 muM) for uptake. Salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, or elevation of cAMP, cGMP, or inhibition of protein kinase C. Salsolinol also inhibited both carbachol (1 mM) and K+ (100 mM, Na+ adjusted) evoked released of noradrenaline from SH-SY5Y cells, with IC50 values of 500 muM and 120 muM, respectively. In conclusion, salsolinol appears to be cytotoxic to SH-SY5Y cells, via a mechanism that does not require uptake 1, bioactivation by monoamine oxidase, or membrane based free radical damage. The effects of salsolinol on catecholamine uptake, and the mechanism of toxicity require further investigation.