Structure of erythrocruorin in different ligand states refined at 1.4 A resolution.

The crystal structure of erythrocruorin has been refined by constrained crystallographic refinement at 1·4 Å resolution in the following ligand states: aquomet (Fe³⁺, high spin), cyanomet (Fe³⁺, low spin), deoxy (Fe²⁺, high spin) and carbonmonoxy (Fe²⁺, low spin). The final R-value at this resolution is better than 0·19 for each of these models. The positional errors of the co-ordinates are less than 0·1 Å.The root-mean-square differences between the deoxygenated and the ligated erythrocruorin are about 0·1 Å, being largest for cyanomet-erythrocruorin. The changes in tertiary structures propagate from the location of primary events and often fade out at the molecular surface. Helix E passing the distal side of the haem group is affected most by the direct contact with the ligand bound to the haem iron.Steric hindrance by the distal residue IleE11 forces the cyanide and carbonmonoxide ligands to bind at an angle to the haem axis. The strain at the ligand is partially relieved by movement of the haem deeper into the haem pocket and rearrangement of neighbouring residues.The differences in iron location with respect to the mean haem plane are spin-dependent but unexpectedly small (the largest value is 0·15 Å between deoxy and carbonmonoxy-erythrocruorin). Spin state changes seem to have little influence on the porphyrin stereochemistry; it is determined primarily by the chemical properties of the ligand and its interaction with the haem and the globin. These non-covalent interactions are largely responsible for the initiation of the structural changes on ligand binding.     

Carvedilol inhibition of lipid peroxidation. A new antioxidative mechanism

To define the molecular mechanism(s) of carvedilol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process.

Carvedilol inhibits the peroxidation of sonicated phosphatidylcholine liposomes triggered by FeCl₂ addition whereas atenolol, pindolol and labetalol are ineffective. The inhibition proved not to be ascribable (a) to an effect on Fe²⁺ autoxidation and thus on the generation of oxygen derived radical initiators; (b) to the scavenging of the inorganic initiators O^·-₂ and ^·OH; (c) to an effect on the reductive cleavage of organic hydroperoxides by FeCl₂; (d) to the scavenging of organic initiators. The observations that (a) carvedilol effectiveness is inversely proportional to the concentration of FeCl₂ and lipid hydroperoxides in the assay; (b) the drug prevents the onset of lipid peroxidation stimulated by FeCl₃ addition and; (c) it can form a complex with Fe³⁺, suggest a molecular mechanism for carvedilol action. It may inhibit lipid peroxidation by binding the Fe³⁺ generated during the oxidation of Fe²⁺ by lipid hydroperoxides in the substrate. The lag time that carvedilol introduces in the peroxidative process would correspond to the time taken for carvedilol to be titrated by Fe³⁺; when the drug is consumed the Fe³⁺ accumulates to reach the critical parameter that stimulates peroxidation. According to this molecular mechanism the antioxidant potency of carvedilol can be ascribed to its ability to bind a species, Fe³⁺, that is a catalyst of the process and to its lipophilic nature that concentrates it in the membranes where Fe³⁺ is generated by a site specific mechanism.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

The effect of buffers and chelators on the reaction of luminol with Fenton's reagent near neutral pH

The chemiluminescence of the system luminol +Fe²⁺ + H₂O₂ was measured in aqueous buffer at pH 7.2. In veronal (5,5-diethybarbiturate) buffer, the luminescence is strongly quenched by ethanol and mannitol, but only weakly by t-butanol, benzoate and superoxide dismutase (SOD); complexing Fe²⁺ with 1,10-phenanthroline or 2,2′-dipyridyl causes a decrease of light production that can be partially obviated by the simultaneous addition of SOD. In phosphate buffer, the luminescence is higher than in veronal and it is efficiently quenched by all four OH · quenchers and by SOD. In Tris buffer, no light production is observed as long as the Fe²⁺ is not complexed. When Fe²⁺ is complexed by pyrophosphate or phytate, there is a strong chemiluminescence in all three buffers, which is quenched by all four OH · quenchers and by SOD. When Fe²⁺ is complexed by EDTA or DTPA, very little luminescence is observed. The luminol analogue phthalhydrazide, which was suggested by Merényi and Lind as a reliable OH · detector, can replace luminol only in phosphate buffer, and thus turns out to be very specific indeed for free OH ·.     

Seasonal patterns of cell-to-cell communication in Chara corallina Klein ex Willd. I. Cell-to-cell communication in vegetative lateral branches during winter and spring

Cell-to-cell communication in lateral branches of limited growth in male Chara corallina plants has been studied using fluorescent plasmalemma-impermeable probes. The extent of cell-to-cell communication was found to be seasonal. In winter, branch internodes were characterized by relatively low plasmalemma potential differences (–121·2±23·2 mV; [K⁺]_o 0·5 mol m^?3; pH 7·6), quiescent dactyls isolated from the symplast (conic tips) and by restricted cell-cell communication (frequency of intercellular transport of six carboxyfluorescein = 42·9%). Cell-to-cell communication was inhibited during action-potentials, and was more extensive between cells which did not give an action potential in response to a current pulse. An inhibitor of the action potential, La³⁺, promoted cell-to-cell communication. These may be characteristic features of winter dormancy in lateral branches of Chara. In spring, morphological and electrophysiological changes occurred immediately prior to the onset of fertility. Characteristic changes in morphology included increased abundance of spherosomes and glycosomes, elongation of stipulodes and development of active branch dactyls. Spring branch inter-nodes were characterized by high plasmalemma PDs (–210·5±30·8mV; [K⁺]_o 0·5 mol m^?3; pH 7·6), predominantly active, non-isolated dactyls (spinaceous tips), and extensive intercellular communication (frequency of intercellular transport of 6 carboxyfluorescein = 89·2%). Artificial elevation of the intracellular calcium ion concentration by application of ionophore A23187 or Ca²⁺ microinjection significantly restricted intercellular communication to a level similar to that found in winter. The depression of intercellular communication in winter tissues is suggested to be due to high sensitivity to action potentials and/or to Ca²⁺ fluxes. Changes in intracellular Ca²⁺ distribution may be involved in the transition between the morphologically distinct states of dormancy and incipient fertility.     

Nutrient status of black spruce (Picea mariana [Mill.] BSP) forest soils dominated by Kalmia angustifolia L.

A study was conducted to determine soil chemistry in an uncut black spruce (Picea mariana) forest with and without the ericaceous understory shrub Kalmia angustifolia, as well as on a cut black spruce forest currently dominated by Kalmia. The organic (humus) and mineral (Ae, upper and lower B horizons) soils associated with Kalmia from uncut and cut forests, and non-Kalmia soils from uncut forest, were analyzed for selected soil properties. In general, mineral soils (B horizon) associated with Kalmia in uncut forest have lower values for organic matter (3.25%), organic nitrogen (0.66 mg·g⁻¹), Fe³⁺ (95.4 μg·g⁻¹) and Mn²⁺ (9 μg·g⁻¹), and higher values for pH (4.12) and Ca²⁺ (27 μg·g⁻¹) compared to non-Kalmia (organic matter, 3.43%; organic-N, 1.15 mg·g⁻¹; Fe³⁺, 431 μg·g⁻¹; Mn²⁺, 23.2 μg·g⁻¹; pH, 3.14; Ca²⁺, 15.6 μg·g⁻¹) and cut black spruce-Kalmia (organic matter, 3.74%; organic-N, 0.94 mg·g⁻¹; Fe³⁺, 379 μg·g⁻¹; Mn²⁺, 27 μg·g⁻¹; pH, 2.87; Ca²⁺, 25.2 μg·g⁻¹) forest. The high C:N ratio in Kalmia mineral soil from upper B (29.73) and lower B (identified as B+) (33.08) in uncut black spruce forest was recorded compared to non-Kalmia soils in B (18.17) and B+ (17.05) horizons in uncut black spruce forest. Phenolics leached out from Kalmia litter were lower in Kalmia associated soils than the non-Kalmia soils from the uncut forest, and Kalmia associated soils from the cut forest area. Results indicate that soils associated with Kalmia were nutrient poor particularly for nitrogen, phosphorus, iron and manganese, and provide some basis for the hypothesis that Kalmia has dominated microsites that were nutrient poor prior to Kalmia colonization.     

Calcium accumulation by chick intestinal mitochondria: Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3

We have evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca²⁺ accumulation by chick intestinal mitochondria. Ca²⁺ accumulation appears to occur in two phases: an early, transient accumulation into an Na⁺-labile pool followed by an ATP-dependent accumulation into an Na⁺-resistant pool. Ca²⁺ accumulation is extensive at free Ca²⁺ concentrations greater than 3 · 10^?6 M in the presence of ATP. Ruthenium red and dinitrophenol block Ca²⁺ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)₂D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca²⁺, or the rate and extent of ATP-supported Ca²⁺ accumulation. Intestinal cytosol stimulated Ca²⁺ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca²⁺-binding protein. 30 ng/ml 1,25(OH)₂D-3 stimulated Ca²⁺ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 > 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca²⁺ concentration from 3 · 10^?6 to 1 · 10^?5 M increased Ca²⁺ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)₂D-3 in vitro increased Ca²⁺ accumulation less than 2-fold, we conclude that 1,25(OH)₂D-3 influences mitochondrial accumulation of Ca²⁺ in vivo primarily by altering cytosol concentrations of free Ca²⁺.     

Regulation of Fe3+-oxide Formation Among Fe2+-oxidizing Bacteria

Helical stalks (resembling Gallionella ferruginea, Mariprofundus ferrooxydans) and filamentous sheaths (resembling Leptothrix ochracea) of Fe²⁺-oxidizing bacteria (FeOB) are mineralized by hydrous ferric oxides (HFO). To perform both inter-species and inter-site size comparisons of HFO particles on stalks and sheaths we measured HFO particles in samples of natural bacteriogenic iron oxides (BIOS) from 3 contrasting field sites: the Loihi Seamount (southern Hawaii); Äspö Hard Rock Laboratory (eastern Sweden); and Chalk River Laboratories (northern Canada) representing seafloor saline, underground brackish, and surface freshwater aqueous conditions. Ambient temperatures were in the psychrophilic range and pHs measured for Loihi, CRL, and Äspö were 5.6, 6.9 and 7.4, respectively. Dissolved Fe was lowest for CRL (0.2 mg · L^?1) followed by Äspö (1.5 mg · L^?1), then Loihi (4.5–14.9 mg · L^?1). L. ochraceasheaths appear to have surface properties that restrict HFO particle growth in comparison to G.ferruginea-M.ferrooxydans stalks in the same environment, which we attribute to interfacial surface energy (γ). An inverse relationship between particle size and stalk/sheath length due to restrictions in reactive surface area was also observed, which may provide insight into FeOB survival strategies to alleviate oxidative stress arising from Fe³⁺ production.     

Decarboxylation of homoarginine and lysine by an enzyme from Lathyrus sativus seedlings

Homoarginine decarboxylase has been purified ca 110-fold from Lathyrus sativus seedlings and resolved from arginine decarboxylase by DEAE-Sephadex column chromatography. The enzyme was less active than arginine decarboxylase and was highly labile. This preparation decarboxylated l-lysine in addition to L-homoarginine. The purified enzyme preparation had an absolute requirement for exogenous Mn²⁺ or Fe²⁺ for both the enzyme activities. The pH and temperature optima for decarboxylation of both homoarginine and lysine were the same viz. 8·4 and 41° respectively. The K_m value l-homoarginine was 3·33 mM and for l-lysine was 0·88 mM. Arginine and homoarginine decarboxylases appear to be different and separable entities having different physico-chemical characteristics, despite the fact that their respective guanido amino acid substrates undergo similar metabolic conversion to guanido- and diamines in this plant system.     

Zinc and salinity effects on membrane transport in Chara connivens

Pressure-probe measurements showed that the pressure relaxation of internodal cells of the freshwater alga Chara connivens slowed considerably when 1–5 mol m^?3 Zn²⁺, or more especially Zn²⁺ and 75 mol m^?3 NaCl, were present in the medium for periods of 1 h or longer. These results indicate that the water permeability of the Chara membrane is decreased by Zn²⁺, and that this effect is enhanced by 75 mol m^?3 NaCl. Specific values taken after 375 min exposure were: 5 mol m^?3 Zn²⁺ and 75 mol m^?3 NaCl caused the half-time for bulk water movement to increase from 7·8±2·3 to 79·5±5·4s, corresponding to a decrease in the hydraulic conductivity (Lp) from (13·0±3·3) × 10^?7 m s^?1 mPa^?1 to (1·25±0·23) × 10^?7 m s^?1 MPa^?1 (mean±S.D., n= 10). These changes are not seen in the presence of NaCl alone, and to a reduced extent in the presence of 5 mol m^?3Zn²⁺ alone (after 375 min, Lp was (2·4±0·1) × 10^?7 m s^?1 MPa^?1, mean±S.D., n = 6). Ca²⁺ cannot substitute for Zn²⁺, but seems to competitively inhibit Zn²⁺. There was another, kinetically distinct effect of Zn²⁺: the ingress of Na⁺ within 15 min of exposure to 75 mol m^?3 NaCl is halved by the presence of 1–5 mol m^?3 Zn²⁺, although internal osmolality is little changed by Zn²⁺. In spite of this, Zn²⁺ does not exert the long-term protection against NaCl that has been reported for Ca²⁺. Depending on the concentration of Zn²⁺ and the duration of the exposure, the effects on water permeability were fully or partly reversible within 24–48 h. The mechanism of these changes is difficult to identify. One possibility is a zinc-induced restriction of trans-membrane channels to give single-file channels which can be blocked by salt.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Ferrous Iron and Sulfur Oxidation and Ferric Iron Reduction Activities of Thiobacillus ferrooxidans Are Affected by Growth on Ferrous Iron, Sulfur, or a Sulfide Ore

Eight strains of Thiobacillus ferrooxidans (laboratory strains Tf-1 [= ATCC 13661] and Tf-2 [= ATCC 19859] and mine isolates SM-1, SM-2, SM-3, SM-4, SM-5, and SM-8) and three strains of Thiobacillus thiooxidans (laboratory strain Tt [= ATCC 8085] and mine isolates SM-6 and SM-7) were grown on ferrous iron (Fe²⁺), elemental sulfur (S⁰), or sulfide ore (Fe, Cu, and Zn). The cells were studied for their aerobic Fe²⁺ - and S⁰-oxidizing activities (O₂ consumption) and anaerobic S⁰-oxidizing activity with ferric iron (Fe³⁺) (Fe²⁺ formation). Fe²⁺-grown T. ferrooxidans cells oxidized S⁰ aerobically at a rate of 2 to 4% of the Fe²⁺ oxidation rate. The rate of anaerobic S⁰ oxidation with Fe³⁺ was equal to the aerobic oxidation rate in SM-1, SM-3, SM-4, and SM-5, but was only one-half or less that in Tf-1, Tf-2, SM-2, and SM-8. Transition from growth on Fe²⁺ to that on S⁰ produced cells with relatively undiminished Fe²⁺ oxidation activities and increased S⁰ oxidation (both aerobic and anaerobic) activities in Tf-2, SM-4, and SM-5, whereas it produced cells with dramatically reduced Fe²⁺ oxidation and anaerobic S⁰ oxidation activities in Tf-1, SM-1, SM-2, SM-3, and SM-8. Growth on ore 1 of metal-leaching Fe²⁺-grown strains and on ore 2 of all Fe²⁺-grown strains resulted in very high yields of cells with high Fe²⁺ and S⁰ oxidation (both aerobic and anaerobic) activities with similar ratios of various activities. Sulfur-grown Tf-2, SM-1, SM-4, SM-6, SM-7, and SM-8 cultures leached metals from ore 3, and Tf-2 and SM-4 cells recovered showed activity ratios similar to those of other ore-grown cells. It is concluded that all the T. ferrooxidans strains studied have the ability to produce cells with Fe²⁺ and S⁰ oxidation and Fe³⁺ reduction activities, but their levels are influenced by growth substrates and strain differences.     

Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C 4 cage axes

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe₂O₃·H₂O minerals from Fe²⁺ for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe²⁺ transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe²⁺/O₂ catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe²⁺ + O₂ protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe₂O₃·H₂O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.     

Serotonin binding protein:enhancement of binding by Fe2+ and inhibition of binding by drugs.

The binding of serotonin to a soluble, high affinity binding protein, present in synaptosomes and associated with serotonergic tracts, has now been studied for the effects of metallic ions and various drugs. At optimal concentration (10^-4 M) of Fe²⁺ the enhancement of binding was close to 20-fold. A much smaller effect was noted with Cu²⁺. With other ions (Fe³⁺, Mn²⁺, Co²⁺, Ni²⁺, Cr³⁺, Mg²⁺, Ca²⁺) little or no effect was seen. For the effect with Fe²⁺. preincubation was required (10 min, 25°C) and concentrations higher than 10^-4M were inhibitory. Studies based on equilibrium dialysis show that the effect of Fe²⁺ was on the affinity of the binding of serotonin to the protein, rather than on the binding capacity. In polydcrylamide gels at pH 8.6 the migratory properties of thc serotonin-protein complex formed in the presence of Fe²⁺ differ from those of the complex formed without Fe²⁺. Nucleotides (ATP, GTP, ADP, AMP) inhibited thc binding. The effects of several classes of drugs (inhibitors of biogenic amine storage and uptake, psychotomimetics, MAO) inhibitors and drugs binding to contractile proteins) were also studied. The only effective inhibitors of serotonin binding were reserpine, vinblastine and CZ-74, which caused 50% inhibition at 2 × 10^-6 M, 7.5 × 10^-6 M and 0.2 × 10^-6M respectively.     

In Vitro Effect of H2O2, Some Transition Metals and Hydroxyl Radical Produced Via Fenton and Fenton-Like Reactions,on the Catalytic Activity of AChE and the Hydrolysis of ACh

It is well known that the principal biomolecules involved in Alzheimer’s disease (AD) are acetylcholinesterase (AChE), acetylcholine (ACh) and the amyloid beta peptide of 42 amino acid residues (Aβ42). ACh plays an important role in human memory and learning, but it is susceptible to hydrolysis by AChE, while the aggregation of Aβ42 forms oligomers and fibrils, which form senile plaques in the brain. The Aβ42 oligomers are able to produce hydrogen peroxide (H₂O₂), which reacts with metals (Fe²⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺) present at high concentrations in the brain of AD patients, generating the hydroxyl radical (^·OH) via Fenton (FR) and Fenton-like (FLR) reactions. This mechanism generates high levels of free radicals and, hence, oxidative stress, which has been correlated with the generation and progression of AD. Therefore, we have studied in vitro how AChE catalytic activity and ACh levels are affected by the presence of metals (Fe³⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺), H₂O₂ (without Aβ42), and ^· OH radicals produced from FR and FLR. The results showed that the H₂O₂ and the metals do not modify the AChE catalytic activity, but the ^·OH radical causes a decrease in it. On the other hand, metals, H₂O₂ and ^·OH radicals, increase the ACh hydrolysis. This finding suggests that when H₂O₂, the metals and the ^·OH radicals are present, both, the AChE catalytic activity and ACh levels diminish. Furthermore, in the future it may be interesting to study whether these effects are observed when H₂O₂ is produced directly from Aβ42.     

Latitudinal and altitudinal growth patterns of brown trout Salmo trutta at different spatial scales

Spatial variation in growth of stream‐dwelling brown trout Salmo trutta was explored in 13 populations using a long‐term study (1993–2004) in the Bay of Biscay drainage, northern Spain. The high variability in fork length (L_F) of S. trutta in the study area was similar to the body‐size range found in the entire European distribution of the species. Mean L_F at age varied: 0+ years, 57·4–100·7 mm; 1+ years, 111·6–176·0 mm; 2+ years, 155·6–248·4 mm and 3+ years, 194·3–290·9 mm. Average L_F at age was higher in main courses and lower reaches compared with small tributaries and upper reaches. Annual specific growth rates (G_L) were: 0+ to 1+ years, 0·634–0·825 mm mm⁻¹ year⁻¹; 1+ to 2+ years, 0·243–0·342 mm mm⁻¹ year⁻¹; 2+ to 3+ years, 0·166–0·222 mm mm⁻¹ year⁻¹, showing a great homogeneity. Regression models showed that water temperature and altitude were the major determinants of L_F at age variability within the study area. A broader spatial analysis using available data from stream‐dwelling S. trutta populations throughout Europe indicated a negative relationship between latitude and L_F of individuals and a negative interaction between latitude and altitude. These findings support previous evidence of the pervasive role of water temperature on the L_F of this species. Altitude appeared as the overall factor that includes the local variation of other variables, such as water temperature or food availability. At a larger scale, latitude was the factor that encompassed these environmental gradients and explained the differences in L_F of S. trutta. In summary, L_F at age in stream‐dwelling S. trutta decreases with latitude in Europe, the converse of Bergmann's rule.     

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N₂L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick‐end labelling (TUNEL)], plasma membrane integrity (SYBR‐14/PI) and mitochondrial membrane potential (ΔΨMMit, JC‐1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10⁷ spermatozoa oocyte^?1, by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d . DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (V_CL) 61·2 ± 17·4 µm s^?1; average‐path velocity (V_AP) 50·1 ± 17·3 µm s^?1; straight‐line velocity (V_SL) 59·1 ± 18·4 µm s^?1; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, V_CL, V_AP and V_SL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, V_CL and V_SL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with V_CL and V_SL (r = 0·59 and r = 0·55, respectively).     

Effects of metal ions on benzylglucosinolate degradation in Lepidium sativum seed autolysates

The effects of varying concentrations of Fe²⁺ (5 × 10^?5 ?5 × 10^?1 M) on benzylglucosinolate degradation in Lepidium sativum seed autolysates were investigated. Increased glucosinolate decomposition was observed over the whole range with a maximum effect at ca 6 × 10^?3 M Fe²⁺, at which point glucosinolate degradation was more than three times that obtained in the absence of added Fe²⁺ . Nitrile formation was especially enhanced in the presence of all concentrations of Fe²⁺ studied, and maximum amounts were obtained at ca 6 × 10^?3 M Fe²⁺ when a more than four-fold increase over quantities produced in the absence of Fe²⁺ was observed. Thiocyanate formation was also promoted with a maximum at ca 4 × 10^?3 M Fe²⁺, but isothiocyanate production was considerably reduced in allcases. It is suggested that Fe²⁺ inhibits isothiocyanate formation by interfering with the availability of ascorbic acid which is a proven co-factor for most thioglucosidase isoenzymes, but that an Fe²⁺-ascorbate complex might then be responsible for promoting enzymic production of nitrile. The effects of a limited range of concentrations of Fe³⁺ and Cu⁺ were also studied, and results related to those for Fe²⁺. The relevance of the findings to natural systems and to glucosinolate-containing foods is briefly discussed.     

In vitro antioxidant activity of silymarin

Silymarin, a known standardized extract obtained from seeds of Silybum marianum is widely used in treatment of several diseases of varying origin. In the present paper, we clarified the antioxidant activity of silymarin by employing various in vitro antioxidant assay such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH·) scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by Fe³⁺ ? Fe²⁺ transformation method and Cuprac assay, superoxide anion radical scavenging by riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe²⁺) chelating activities. Silymarin inhibited 82.7% lipid peroxidation of linoleic acid emulsion at 30 μg/mL concentration; butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox indicated inhibition of 83.3, 82.1, 68.1 and 81.3% on peroxidation of linoleic acid emulsion at the same concentration, respectively. In addition, silymarin had an effective DPPH· scavenging, ABTS^√+ scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe³⁺) reducing power by Fe³⁺ ? Fe²⁺ transformation, cupric ions (Cu²⁺) reducing ability by Cuprac method, and ferrous ions (Fe²⁺) chelating activities. Also, BHA, BHT, α-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. Moreover, this study, which clarifies antioxidant mechanism of silymarin, brings new information on the antioxidant properties of silymarin. According to the present study, silymarin had effective in vitro antioxidant and radical scavenging activity. It could be used in the pharmacological and food industry because of its antioxidant properties.