Factors affecting lipid peroxidation in guinea-pig adrenal microsomes

Studies were carried out to examine the effects of and interactions between NADPH, Fe²⁺, Fe³⁺ and ascorbate on lipid peroxidation in guinea-pig adrenal microsomes. Fe²⁺, at levels between 10⁻⁶ and 10⁻³ M, produced concentration-dependent increases in lipid peroxidation in adrenal microsomes; Fe²⁺ had a far greater effect than Fe³⁺. In liver microsomes, by contrast, Fe²⁺ and Fe³⁺ had quantitatively similar effects on lipid peroxidation. NADPH alone had no effect on malonaldehyde production by adrenal microsomes. However, in the presence of low Fe²⁺ concentrations (10⁻⁶ M), NADPH stimulated malonaldehyde production; the stimulation was not demonstrable in microsomes which had been heat-treated to inactive microsomal enzymes. In the presence of high Fe²⁺ levels (10⁻³ M), NADPH produced a concentration-dependent inhibition of lipid peroxidation; the inhibition was fully demonstrable in heat-treated microsomes. In the presence of Fe³⁺ (10⁻⁶ to 10⁻³ M), NADPH had little effect on lipid peroxidation, suggesting that NADPH does not significantly promote the reduction of Fe³⁺ to Fe²⁺ in adrenal microsomes. Ascorbate alone increased malonaldehyde production by adrenal microsomes; maximum stimulation occurred at a concentration of 10⁻⁴ M. Ascorbate-induced lipid peroxidation was also inhibited by NADPH. Ascorbate (5 · 10⁻⁶ to 1 · 10⁻⁴ M) synergistically interacted with low levels (10⁻⁶ M) of Fe²⁺ to enhance malonaldehyde production by adrenal microsomes. The synergism was not demonstrable at high concentrations (10⁻³ M) of Fe²⁺ At all concentrations (10⁻⁶ to 10⁻³ M) of Fe³⁺ studied, ascorbate synergistically increased the production of malonaldehyde. The results indicate that interactions between various endogenous substances may be important in the control of adrenal microsomal lipid peroxidation and that there are differences in the regulation of adrenal and hepatic lipid peroxidation.     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.     

The multiple effects of ethylenediaminetetraacetate in several model lipid peroxidation systems

Experiments were performed which illustrate the various ways EDTA can influence lipid peroxidation. Either detergent-dispersed linoleate, or liposomes made from extracted microsomal phospholipids were utilized as substrates for peroxidation. Peroxidation was accomplished using Fe²⁺ or Fe³⁺. In systems utilizing Fe²⁺, EDTA chelation facilitated Fe²⁺ autoxidation which in turn caused peroxidation of detergent-dispersed linoleate. Peroxidation was not initiated during EDTA-Fe²⁺ autoxidation when the substrate lipids were in a liposomal configuration. Systems utilizing Fe³⁺ required an enzyme (either xanthine oxidase or NADPH-cytochrome P₄₅₀ reductase) to reduce the iron for peroxidative activity. EDTA chelation of Fe³⁺ enhanced the xanthine oxidase and NADPH-cytochrome P₄₅₀ reductase-catalyzed peroxidation of detergent-dispersed linoleate, presumably by facilitating the reduction of Fe³⁺. Catalase and mannitol inhibited both EDTA-Fe²⁺- and EDTA-Fe³⁺-dependent lipid peroxidation. EDTA-Fe³⁺ was not capable of initiating peroxidation of phospholipid liposomes following enzymatic reduction by either enzyme, but ADP-chelated iron effectively initiated liposomal peroxidation in similar systems. With xanthine oxidase-catalyzed peroxidation of liposomes with ADP-Fe³⁺, the inclusion of EDTA-Fe³⁺ caused a modest enhancement of activity. EDTA-Fe³⁺ greatly stimulated NADPH-cytochrome P₄₅₀ reductase-catalyzed peroxidation of liposomes with ADP-Fe³⁺. In contrast, the addition of EDTA, rather than EDTA-Fe³⁺ inhibited the liposomal peroxidation catalyzed by either enzyme with ADP-Fe³⁺ when the EDTA concentration exceeded the concentration of Fe³⁺.     

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.     

Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which may result from activation of Ca²⁺ permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca²⁺]_i from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca²⁺ channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30–48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37°C) significantly increased glycation of membrane proteins, hemoglobin (HbA_1c), TRPC3/6/7, and L-type Ca²⁺ channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca²⁺]_i, and phosphatidylserine exposure, and led to significant cell shrinkage. Ca²⁺ removal and addition of Ca²⁺ chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca²⁺ entry from extracellular space.     

Fe and Fe initiated peroxidation of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media

Retina is highly susceptible to oxidative damage due to its high content of polyunsaturated fatty acids (PUFAs), mainly docosahexaenoic acid (22:6 n3). Lipid peroxidation process is thought to be involved in many physiological and pathological events. Many model membranes can be used to learn more about issues that cannot be studied in biological membranes. Sonicated liposomes (SL) and non-sonicated liposomes (NSL) prepared with lipids isolated from bovine retina and characterized by dynamic light-scattering, were submitted to lipid peroxidation, under air atmosphere at 22 °C, with Fe²⁺ or Fe³⁺ as initiator, in different aqueous media. Conjugated dienes and trienes, determined by absorption at 234 and 270 nm respectively, and thiobarbituric acid-reactive substances were measured as a function of time. Peroxidation of SL or NSL initiated with 25 μM FeSO₄ in 20 mM Tris-HCl pH 7.4 resulted in an increase in TBARS production after a lag phase of 60 min. Incubation of both types of liposomes in water resulted in shortening of the lag phase at 30 min. When lipid peroxidation was performed in 0.15 M NaCl, lag phase completely disappeared. On the other hand, FeCl₃ (25 μM) induced a limited production of TBARS only just after 30 min of incubation. When Fe²⁺- or Fe³⁺-lipid peroxidation of both types of liposomes was carried out in water or 0.15 M NaCl, formation of conjugated dienes and conjugated trienes were higher than in reactions carried out in 20 mM Tris-HCl pH 7.4.Our results established that both liposome types were susceptible to Fe²⁺- and Fe³⁺-initiated lipid peroxidation. However, Fe²⁺ showed a clearly enhanced effect on peroxidation rate and steady state concentration of oxidation products.We verified that peroxidation of liposomes made of retinal lipids is affected not only by type of initiator but also by aqueous media. This model constitutes a useful system to study formation of lipid peroxidation intermediaries and products in an aqueous environment.     

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe²⁺) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n = 13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe²⁺, EE-CAT plus Fe²⁺, EE-GPx plus Fe²⁺ and EE-SOD plus Fe²⁺). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6 h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6 h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2 h after thawing (P < 0.05). Catalase supplementation, however, improved DNA integrity at 4 h (P < 0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6 h, linear motility at 6 h, mitochondrial activity at 6 h, membrane integrity at 2 and 6 h, and DNA integrity at 4 h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2 h after thawing (P < 0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2 h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe²⁺ negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P < 0.05). After thawing, there were, however, no significant differences between the control plus Fe²⁺ and the antioxidative enzymes supplementation plus Fe²⁺ groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.     

Effects of lipid peroxidation on adrenal microsomal monooxygenases

Incubation of guinea pig adrenal microsomes with 10^?6 M ferrous (Fe²⁺) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe²⁺ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe²⁺ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[α]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.     

Clofibric acid or diethylmaleate supplemented diet decrease blood pressure in DOCA-salt treated male Sprague-Dawley rats - relation with liver antioxidant status

The effects of ascorbate and a-tocopherol as antioxidants and as co-operative factors against NADPH-dependent lipid peroxidation in human placental mitochondria have been studied. The addition of ascorbate at low concentration (up to 50 M) to the NADPH-generating system resulted in increasing lipid peroxidation and Fe³⁺ to Fe²⁺ reduction. High concentration of ascorbate (150 M), which produced maximal rate of ascorbate-dependent lipid peroxidation, was found to inhibit almost completely NADPH-dependent lipid peroxidation by maintaining too much iron in its reduced form. Either stimulatory or inhibitory effect of ascorbate on NADPH-dependent lipid peroxidation depends on the appropriate Fe³⁺/Fe²⁺ ratio. -Tocopherol caused a decrease of NADPH-dependent lipid peroxidation, inhibiting completely this process at 150 M concentration. The inhibitory effect of -tocopherol increased rapidly with the increasing ascorbate concentration, almost complete inhibition of NADPH-dependent lipid peroxidation being obtained at 25 M -tocopherol and 50 M ascorbate. This strong inhibitory combined effect of -tocopherol and ascorbate was independent of the Fe³⁺/Fe²⁺ ratio, as a-tocopherol is not able to reduce Fe³⁺ to Fe²⁺ under the conditions employed. These findings suggest that antioxidant effects of ascorbate in placental mitochondria are mediated by recycling of a-tocopherol rather than by strong reduction of Fe³⁺ to Fe²⁺. On the basis of the results obtained, we assume that adequate concentrations of a-tocopherol and ascorbate in placental tissue may prevent the release of lipid peroxide from placental mitochondria and therefore could be protective against the development of preeclampsia.     

Physiological significance of 3-h bright and dim light exposure prior to taking a bath for core and forehead skin temperatures and heart rate during 1-h bathing of 38.5°C

1. The study investigated the effect of exposure to 3-h bright light (2500 lx) or dim light (200 lx) just prior to taking a hot bath upon thermophysiological responses during the 1-h bath (at 38.5°C water temperature). 2. Core and forehead skin temperature increases during the bath were significantly lower after bright than after dim light exposure. 3. Heart rate during the bath was significantly lower after exposure to bright light than dim light. 4. These results are discussed in terms of a reduced set-point of core temperature due to a probable higher secretion of melatonin under the bright light condition.     

Effects of iron-induced lipid peroxidation and of acidosis on choline uptake by synaptosomes

The effects of iron-induced lipid peroxidation and of lactic acidosis on [³H]choline uptake were investigated on crude synaptosomes prepared from rat cerebral cortices. Fe²⁺-induced lipid peroxidation as evidenced from the production of thiobarbituric acid reactives substances (TBARS) was correlated with a decrease in high-affinity choline uptake (HACU). Trolox C, a free radical scavenger, prevented both Fe²⁺-induced TBARS production and decrease in HACU. Lactic acidosis (pH 6.0 for 30 or 60 min) increased the TBARS production with concomitant decrease in HACU (–48%, –78%, respectively). The acidosis dependent decrease was not reversible following pH 7.4 readjustment after 60 min acidosis. It was not prevented by trolox C, although trolox C inhibited the acidosis-induced production of TBARS. The results suggest that the contribution of acidosis to peroxidative damages is probably of less importance in comparison to other cytotoxic mechanisms.     

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.     

Solubilization of adenylate cyclase of brain membranes by lipid peroxidation

Adenylate cyclase in the membrane fractions of bovine and rat brains, but not in rat liver plasma membranes, was solubilized by treatment with Fe²⁺ (10 μM) plus dithiothreitol (5 mM). Solubilization of the enzyme by these agents was completely prevented by simultaneous addition of N,N′-diphenyl-p-phenylenediamine (DPPD), an inhibitor of lipid peroxidation. Ascorbic acid also solubilized the enzyme from the brain membranes. Lipid peroxidation of the brain membranes was characterized by a selective loss of phosphatidylethanolamine. Solubilization of membrane-bound enzymes by Fe²⁺ plus dithiothreitol was not specific for adenylate cyclase, because phosphodiesterase, thiaminediphosphatase and many other proteins were also solubilized. Solubilized adenylate cyclase had a high specific activity and was not activated by either NaF, 5′-guanylyl imidodiphosphate (Gpp[NH]p) or calmodulin. These results suggested that lipid peroxidation of the brain membranes significantly solubilized adenylate cyclase of high specific activity.     

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes.

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes. This reaction was heat-insensitive contrary to the mitochondrial peroxidation reported in the previous paper, and was enhanced by p-chloromercuribenzoate. Additions of Fe²⁺ and Fe³⁺ stimulated both the lipid peroxidation and the disappearance of dihydroxyfumaric acid. On the other hand, addition of Mn²⁺ or Cu²⁺, which stimulated the disappearance of dihydroxyfumaric acid, inhibited the lipid peroxidation. Hydroxyl radical scavengers, superoxide dismutase and catalase had no effect on this lipid peroxidation and dihydroxyfumaric acid disappearance. The cytochrome p-450 content decreased about 70 % in parallel with the lipid peroxidation.     

Linoleic acid peroxidation initiated by Fe-reducing compounds recovered from Eucalyptus grandis biotreated with Ceriporiopsis subvermispora

This work evaluates linoleic acid peroxidation reactions initiated by Fe³⁺-reducing compounds recovered from Eucalyptus grandis, biotreated with the biopulping fungus Ceriporiopsis subvermispora. The aqueous extracts from biotreated wood had the ability to reduce Fe³⁺ ions from freshly prepared solutions. The compounds responsible for the Fe³⁺-reducing activity corresponded to UV-absorbing substances with apparent molar masses from 3 kDa to 5 kDa. Linoleic acid peroxidation reactions conducted in the presence of Fe³⁺ ions and the Fe³⁺-reducing compounds showed that the rate of O₂ consumption during peroxidation was proportional to the Fe³⁺-reducing activity present in each extract obtained from biotreated wood. This peroxidation reaction was coupled with in-vitro treatment of ball-milled E. grandis wood. Ultraviolet data showed that the reaction system released lignin fragments from the milled wood. Size exclusion chromatography data indicated that the solubilized material contained a minor fraction representing high-molar-mass molecules excluded by the column and a main low-molar-mass peak. Overall evaluation of the data suggested that the Fe³⁺-reducing compounds formed during wood biodegradation by C. subvermispora can mediate lignin degradation through linoleic acid peroxidation.     

Two light sources differentially affected ferric iron reduction and growth of cotton

In growth chambers, low pressure sodium (LPS) plus incandescent (Inc) lamps and fluorescent cool-white (FCW) plus Inc lamps were used to determine their effects on growth of cotton (Gossypium hirsutum L.) and on the reduction of Fe³⁺ to Fe²⁺. Cotton plants grown under LPS + Inc light developed chlorosis and grew poorly, whereas plants grown under FCW + Inc lights were green. The chlorophyll concentration and top and root weights of cotton grown under LPS + Inc were lower than those under FCW + Inc. In solution, FCW + Inc lamps reduced about eight times more Fe³⁺ to Fe²⁺ than did LPS + Inc lamps. Fe³⁺ is transported to plant tops as Fe³⁺ citrate and if we assume that FCW + Inc light reduces Fe³⁺ to Fe²⁺ in plant foliage as it did in the solutions, then reduction of Fe³⁺ by the light environment will make Fe²⁺ in the tops more available for biochemical reactions.     

Circadian rhythms and the induction of flowering in the long-day grass Lolium temulentum L.

Plants of Lolium temulentum L. strain Ceres were grown in 8-h short day (SD) for 45 d before being exposed either to a single long day (LD) or to a single 8-h SD given during an extended dark period. For LD induction, the critical photoperiod was between 12 and 14 h, and more than 16 h were needed for a maximal flowering response. During exposure to a single 24-h LD, the translocation of the floral stimulus began between the fourteenth and the sixteenth hours after the start of the light period, and was completed by the twenty-fourth hour. Full flowering was also induced by one 8-h SD beginning 4 or 28 h after the start of a 40-h dark period, i.e. by shifting 12 h forward or beyond the usual SD. The effectiveness of a so-called ‘displaced short day’ (DSD) was not affected by light quality and light intensity. With a mixture of incandescent and fluorescent lights at a total photosynthetic photon flux density of 400 μmol m⁻² s⁻¹, a 4-h light exposure beginning 4 h after the start of a 40-h dark period was sufficient to induce 100% flowering. The flower-inducing effect of a single 8-h DSD was also assessed during a 64-h dark period. Results revealed two maxima at a 20-h interval. This fluctuation in light sensitivity suggests that a circadian rhythm is involved in the control of flowering of L. temulentum.     

Effect of light intensity on Opisthorchis viverrini cercarial shedding levels from Bithynia snails--a preliminary study

Opisthorchis viverrini requires Bithynia snails as the first intermediate host and cyprinid fish as the second intermediate host. Very low natural infection rates have been reported in Bithynia snails, but very high rates have been found in cyprinid fish in the same endemic region. This study investigated the effect of light intensity, the most important stimulus, on the quantity of O. viverrini cercariae shed from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos snails. Snails were evaluated for cercariae output every hour after exposure to various light intensities for a total period of 7 h. The same infected snail was tested under different intensities of light: in the dark, and at 1000, 3000 and 5000 lx. The data showed that under exposure to 1000 and 3000 lx of light, the average percentage and number of cercariae released were higher than that exposed to 5000 lx during the first 2 h of the experiment. In contrast, under higher illumination (5000 lx) a longer time (6 h) was required to stimulate the peak emergence of cercariae. Darkness was not able to induce O. viverrini cercariae emergence. Among the three intensities of light, exposure at 1000 lx induced the highest average number of released cercariae per snail and the highest percentage of cercarial emergence within the first 2 h (125, 54.86%), followed by exposure at 3000 lx (69, 25.58%) and 5000 lx (12, 7.78%). The results suggest that the light intensity of 1000 lx for 2 h would be optimal for O. viverrini cercarial shedding from naturally infected B. (D.) siamensis goniomphalos snails.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe²⁺ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than α-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than α-tocopherol; (e) to be a weaker antiradical than α-tocopherol in the reduction of the stable radical DPPH^·. Resveratrol reduced Fe³⁺ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe³⁺, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like α-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.