Succinate is the controller of O<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at mitochondrial complex I : negative modulation by malate,positive by cyanide

Mitochondrial production of H₂O₂ is low with NAD substrates (glutamate/pyruvate, 3 and 2 mM) (G/P) and increases over ten times upon further addition of succinate, with the formation of a sigmoidal curve (semimaximal value at 290 μM, maximal H₂O₂ production at 600 μM succinate). Malate counteracts rapidly the succinate induced increased H₂O₂ release and moves the succinate dependent H₂O₂ production curve to the right. Nitric oxide (NO) and carbon monoxide (CO) are cytochrome c oxidase inhibitors which increase mitochondrial ROS production. Cyanide (CN⁻) was used to mimic NO and CO. In the presence of G/P and succinate (300 μM), CN⁻ progressively increased the H₂O₂ release rate, starting at 1.5 μM. The succinate dependent H₂O₂ production curve was moved to the left by 30 μM CN⁻. The V_max was little modified. We conclude that succinate is the controller of mitochondrial H₂O₂ production, modulated by malate and CN⁻. We propose that succinate promotes an interaction between Complex II and Complex I, which activates O₂⁻ production.     

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Summary.  Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H⁺ efflux rate, 1.80 μM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H⁺ consumption for O₂ ^•− dismutation to H₂O₂. Also K⁺ influx was strongly depressed by MeJA, even transitorily reverting to K⁺ efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H₂O₂ accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O₂ uptake by roots gave similar results. These and other results for additions of H₂O₂ or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H₂O₂ being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN⁻- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O₂ ^•− and H₂O₂, O₂ uptake, and peroxidase activity by roots. Received July 12, 2002; accepted October 2, 2002; published online May 21, 2003 RID="*"     

Pesticides induced oxidative stress in thymocytes

The role of oxidative stress in immune cell toxicity caused by the pesticides lindane, malathion and permethrin was investigated in thymic cells from C57BL/6 mice. Thymocytes treated with any of these pesticides (concentrations ranging between 50–150 μM) were found to generate both superoxide (^•O₂ ⁻) and H₂O₂. The production of ^•O₂ ⁻ was detected with hydroethidine-ethidium bromide assay. H₂O₂ production was monitored with a flow cytometric fluorescent (DCFH-DA) assay. All three pesticides stimulated ^•O₂ ⁻ release after 5 min exposure. Lindane and permethrin, but not malathion, continued to have significant (p ≤ 0.05) effects on ^•O₂ ⁻ generation following 15 min of exposure. The lindane + malathion mixture was found to cause more-than-additive increase in ^•O₂ ⁻ production compared to individual pesticide treatments (at both 5 and 15 min). However, the effect of the lindane + permethrin mixture was not significantly different than individual components of this mixture. The effects of these pesticides on levels of antioxidant enzymes were also investigated, and only mixtures were found to have significant (p ≤ 0.05) effects. Thus, lindane + malathion and lindane + permethrin mixtures increased total superoxide dismutase (SOD) specific activity, had no effect on catalase levels and inhibited GSH-peroxidase and GSH-reductase specific activities. Although the results of these studies do not explain the mechanism of action of these pesticides on the generation of ^•O₂ ⁻ and H₂O₂, it is worthy of note that mixtures of these chemicals have oxidative responses greater than those of single chemicals. An erratum to this article can be found at     

Astaxanthin ameliorates the redox imbalance in lymphocytes of experimental diabetic rats

Diabetes mellitus is a syndrome of impaired insulin secretion/sensitivity and frequently diagnosed by hyperglycemia, lipid abnormalities, and vascular complications. The diabetic ‘glucolipotoxicity’ also induces immunodepression in patients by redox impairment of immune cells. Astaxanthin (ASTA) is a pinkish-orange carotenoid found in many marine foods (e.g. shrimp, crabs, salmon), which has powerful antioxidant, photoprotective, antitumor, and cardioprotective properties. Aiming for an antioxidant therapy against diabetic immunodepression, we here tested the ability of prophylactic ASTA supplementation (30 days, 20 mg ASTA/kg BW) to oppose the redox impairment observed in isolated lymphocytes from alloxan-induced diabetic Wistar rats. The redox status of lymphocytes were thoroughly screened by measuring: (i) production of superoxide (O₂^?), nitric oxide (NO), and hydrogen peroxide (H₂O₂); (ii) cytosolic Ca²⁺; (iii) indexes of oxidative injury; and (iv) activities of major antioxidant enzymes. Hypolipidemic and antioxidant effects of ASTA in plasma of ASTA-fed/diabetic rats were apparently reflected in the circulating lymphocytes, since lower activities of catalase, restored ratio between glutathione peroxidase and glutathione reductase activities and lower scores of lipid oxidation were concomitantly measured in those immune cells. Noteworthy, lower production of NO and O₂^? (precursors of peroxynitrite), and lower cytosolic Ca²⁺ indicate a hypothetical antiapoptotic effect of ASTA in diabetic lymphocytes. However, questions are still open regarding the proper ASTA supplementation dose needed to balance efficient antioxidant protection and essential NO/H₂O₂-mediated proliferative capacities of diabetic lymphocytes.     

A theoretical study on the reaction mechanism of O<Subscript>2</Subscript> with C<Subscript>4</Subscript>H<Subscript>9</Subscript>• radical

Ab initio calculations have been performed using the complete basis set model (CBS-QB3) to study the reaction mechanism of butane radical (C₄H₉•) with oxygen (O₂). On the calculated potential energy surface, the addition of O₂ to C₄H₉• forms three intermediates barrierlessly, which can undergo subsequent isomerization or decomposition reaction leading to various products: HOO• + C₄H₈, C₂H₅• + CH₂CHOOH, OH• + C₃H₇CHO, OH• + cycle-C₄H₈O, CH₃• + CH₃CHCHOOH, CH₂OOH• + C₃H₆. Five pathways are supposed in this study. After taking into account the reaction barrier and enthalpy, the most possible reaction pathway is C₄H₉• + O₂ → IM1 → TS5 → IM3 → TS6 → IM4 → TS7 → OH• + cycle-C₄H₈O.     

Antioxidant effect of a phytoestrogen equol on cultured muscle cells of embryonic broilers

Previous studies have shown that the in ovo injection of equol can markedly improve the water-holding capacity of muscles of broilers chickens at 7 wk of age through promotion of the antioxidant status. We aimed to investigate directly the antioxidant effects of equol on muscle cells in broilers. Muscle cells were separated from leg muscle of embryos on the 11th day of incubation and treated with equol and H₂O₂, either alone or together. Cells were pretreated with medium containing 1, 10, or 100 μM equol for 1 h prior to the addition of 1 mM H₂O₂ for a further 1 h. Photomicrographs of cells were obtained. Cell viability, malondialdehyde (MDA) content, and L-lactate dehydrogenase (LDH) activity in the cell supernatant, as well as intracellular total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities were determined. Treatment with 1 mM H₂O₂ caused serious damage to cells, indicated by comets with no clear head region but a very apparent tail of DNA fragments. Pretreatment with low (1 μM) but not high concentrations of equol (10 μM) inhibited cell damage, while 100 μM equol caused more serious damage than H₂O₂ alone. Pretreatment with 1 μM equol had no effect on cell viability, while pretreatment with 10 and 100 μM equol significantly decreased cell viability in a dose-dependent manner. Compared with H₂O₂ alone, pretreatment with low-dosage equol markedly decreased LDH activity and MDA production in the supernatant, significantly increased intracellular T-SOD activity (P < 0.05) and tended to increase intracellular GSH-Px activity (0.05 < P < 0.1). Pretreatment with high-dosage equol (10 and 100 μM) significantly enhanced LDH activity, but had no effect on MDA content, T-SOD or GSH-Px activity induced by H₂O_2, except for an obvious increase in GSH-Px activity caused by 10 μM equol. These results indicate that equol at low dosage can prevent skeletal muscle cell damage induced by H₂O₂, while pretreatment with high-dosage equol shows a synergistic effect with H₂O₂ in inducing cell damage.     

Cadmium-induced early changes in O2 •−, H2O2 and antioxidative enzymes in soybean (Glycine max L.) leaves

Cadmium-induced initial changes in the production of reactive oxygen species (ROS) and antioxidant mechanism were investigated in soybean (Glycine max L. cv. Don Mario 4800 RR) leaves. Whole plants (WP) and plants without roots (PWR) were exposed to 0.0, 10.0 and 40.0 μM Cd for 0, 4, 6 and 24 h. Compared to PWR, a higher level of endogenous Cd in WP was associated with a lower oxidative stress measured in terms of lipid peroxidation. Furthermore, O₂ ^•− content decreased in the leaves of Cd-treated WP, whereas it increased in those of Cd-treated PWR. Although O₂ ^•− accumulation in PWR was associated with a decrease in superoxide dismutase (SOD) activity, O₂ ^•− diminution in WP leaves was not related to any increase in SOD activity. H₂O₂ content increased in the leaves of both Cd-treated WP and PWR, and it was concomitant with a corresponding decline in catalase (CAT) and ascorbate peroxidase (APX) activities. When diphenyl iodonium (DPI), an inhibitor of NADPH oxidase, was added, H₂O₂ content remained unchanged in Cd-treated WP, suggesting that NADPH oxidase does not participate in the early hours of Cd toxicity. Taken together, our results showed that early ROS evolution and oxidative damage were different in WP and PWR. This suggests that the response in soybean leaves during the early hours of Cd toxicity is probably modulated by the root.     

Evaluation of antigenotoxicity effects of umbelliprenin on human peripheral lymphocytes exposed to oxidative stress

The protective properties of a prenylated coumarin, umbelliprenin (UMB), on the human lymphocytes DNA lesions were tested. Lymphocytes were isolated from blood samples taken from healthy volunteers. DNA breaks and resistance to H₂O₂-induced damage were measured using a single-cell microgel electrophoresis technique under alkaline conditions (comet assay). Human lymphocytes were incubated in UMB (10, 25, 50, 100, 200, and 400 μM) alone or a combination of different concentrations of UMB (10, 25, 50, 100, 200, and 400 μM) and 25 μM H₂O₂. Untreated cells, ascorbic acid (AA; 25, 50, 100, 200, and 400 μM) and H₂O₂ (25 μM) were considered as negative control, positive control, and the standard antioxidant agent for our study, respectively. Single cells were analyzed with “TriTek Cometscore version 1.5” software. The DNA damage was expressed as percent tail DNA. UMB exhibited a concentration-dependent increase in protection activity against DNA damage induced by 25 μM H₂O₂ (from 67.28% to 39.17%). The antigenotoxic activity of AA, in the range 0–50 μM, was greater than that of UMB. However, no significant difference (p > 0.05) in the protective activity was found between UMB and AA at concentrations of approximately higher than 50 μM.     

Cinnamtannin B-1, a natural antioxidant that reduces the effects of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on CCK-8-evoked responses in mouse pancreatic acinar cells

This work was designed in order to gain an insight on the mechanisms by which antioxidants prevent pancreatic disorders. We have examined the properties of cinnamtannin B-1, which belongs to the class of polyphenols, against the effect of hydrogen peroxide (H₂O₂) in mouse pancreatic acinar cells. We have studied Ca²⁺ mobilization, oxidative state, amylase secretion, and cell viability of cells treated with cinnamtannin B-1 in the presence of various concentrations of H₂O₂. We found that H₂O₂ (0.1–100 μM) increased CM-H₂DCFDA-derived fluorescence, reflecting an increase in oxidation. Cinnamtannin B-1 (10 μM) reduced H₂O₂-induced oxidation of CM-H₂DCFDA. CCK-8 induced oxidation of CM-H₂DCFDA in a similar way to low micromolar concentrations of H₂O₂, and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H₂O₂ induced a slow and progressive increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_c). Cinnamtannin B-1 reduced the effect of H₂O₂ on [Ca²⁺]_c, but only at the lower concentrations of the oxidant. H₂O₂ inhibited amylase secretion in response to cholecystokinin, and cinnamtannin B-1 reduced the inhibitory action of H₂O₂ on enzyme secretion. Finally, H₂O₂ reduced cell viability, and the antioxidant protected acinar cells against H₂O₂. In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca²⁺ overload and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that mediates pancreatitis. Our results support the beneficial effect of natural antioxidants in the therapy against oxidative stress-derived deleterious effects on cellular physiology.     

Red cabbage anthocyanins may protect blood plasma proteins and lipids

The present in vitro study was designed to examine the antioxidative activity of red cabbage anthocyanins (ATH) in the protection of blood plasma proteins and lipids against damage induced by oxidative stress. Fresh leaves of red cabbage were extracted with a mixture of methanol/distilled water/0.01% HCl (MeOH/H₂O/HCl, 50/50/1, v/v/w). Total ATH concentration [μM] was determined with cyanidin 3-glucoside as a standard. Phenolic profiles in the crude red cabbage extract were determined using the HPLC method. Plasma samples were exposed to 100 μM peroxynitrite (ONOO⁻) or 2 mM hydrogen peroxide (H₂O₂) in the presence/absence of ATH extract (5–15 μM); oxidative alterations were then assessed. Pre-incubation of plasma with ATH extract partly reduced oxidative stress in plasma proteins and lipids. Dose-dependent reduction of both ONOO⁻ and H₂O₂-mediated plasma protein carbonylation was observed. ATH extract partly inhibited the nitrative action of ONOO⁻, and significantly decreased plasma lipid peroxidation caused by ONOO⁻ or H₂O₂. Our results demonstrate that anthocyanins present in red cabbage have inhibitory effects on ONOO⁻ and H₂O₂-induced oxidative stress in blood plasma components. We suggest that red cabbage ATH, as dietary antioxidants, should be considered as potentially usable nutraceuticals in the prevention of oxidative stress-related diseases.     

The allelopathy and allelopathic mechanism of phenolic acids on toxic <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Cyanobacterial contamination of water has been a serious problem in recent years. Thus, the effective control of undesired cyanobacteria has become an urgent issue. We studied therefore the effects of ρ-coumaric acid and vanillic acid on toxic Microcystis aeruginosa and the allelopathic mechanisms. The results showed that the growth of toxic M. aeruginosa was significantly inhibited by ρ-coumaric acid and vanillic acid, with an EC₅₀ of 0.26 ± 0.07 and 0.34 ± 0.05 mmol L⁻¹, respectively. Our data also demonstrated that both ρ-coumaric acid and vanillic acid triggered the generation of superoxide anion radicals (O₂ ^•−). The O₂ ^•− might induce a lipid peroxidation which may change cell membrane penetrability, thereby leading to the eventual death of M. aeruginosa. Our current studies further provide evidence that some phenolic acids such as ρ-coumaric acid and vanillic acid may be a potential effective solution for aquatic management.     

Oxygen exposure increases resistance of Desulfovibrio vulgaris Hildenborough to killing by hydrogen peroxide

Inactivation of PerR by oxidative stress and a corresponding increase in expression of the perR regulon genes is part of the oxidative stress defense in a variety of anaerobic bacteria. Diluted anaerobic, nearly sulfide-free cultures of mutant and wild-type Desulfovibrio vulgaris (10⁵–10⁶ colony-forming units/ml) were treated with 0 to 2,500 μM H₂O₂ for only 5 min to prevent readjustment of gene expression. Survivors were then scored by plating. The wild type and perR mutant had 50% survival at 58 and 269 μM H₂O₂, respectively, indicating the latter to be 4.6-fold more resistant to killing by H₂O₂ under these conditions. Significantly increased resistance of the wild type (38-fold; 50% killing at 2188 μM H₂O₂) was observed if cells were pretreated with full air for 30 min, conditions that did not affect cell viability. The resistance of the perR mutant increased less (4.6-fold; 50% killing at 1230 μM H₂O₂), when similarly pretreated. Interestingly, no increased resistance of either was achieved by exposure with 10.6 μM H₂O₂ for 30 min, the highest concentration that could be used without killing the cells. Hence, in environments with low D. vulgaris biomass only the presence of external O₂ effectively activates the perR regulon. As a result, mutant strains lacking one of the perR regulon genes ahpC, dvu0772, rbr1 or rbr2 displayed decreased resistance to H₂O₂ stress only following pretreatment with air.     

Evaluation of endogenous acidic metabolic products associated with carbohydrate metabolism in tumor cells

Tumor cells have a high tolerance for acidic and hypoxic microenvironments, also producing abundant lactic acid through accelerated glycolysis in the presence or absence of O₂. While the accumulation of lactate is thought to be a major contributor to the reduction of pH-circumscribing aggressive tumors, it is not known if other endogenous metabolic products contribute this acidity. Furthermore, anaerobic metabolism in cancer cells bears similarity to homo-fermentative lactic acid bacteria, however very little is known about an alternative pathway that may drive adenosine triphosphate (ATP) production independent of glycolysis. In this study, we quantify over 40 end-products (amines, acids, alcohols, aldehydes, or ketones) produced by malignant neuroblastoma under accelerated glycolysis (+glucose (GLU) supply 1–10 mM) ± mitochondrial toxin; 1-methyl-4-phenylpyridinium (MPP⁺) to abate aerobic respiration to delineate differences between anaerobic vs. aerobic cell required metabolic pathways. The data show that an acceleration of anaerobic glycolysis prompts an expected reduction in extracellular pH (pH_ex) from neutral to 6.7 ± 0.006. Diverse metabolic acids associated with this drop in acidity were quantified by ionic exchange liquid chromatography (LC), showing concomitant rise in lactate (Ctrls 7.5 ± 0.5 mM; +GLU 12.35 ± 1.3 mM; +GLU + MPP 18.1 ± 1.8 mM), acetate (Ctrl 0.84 ± 0.13 mM: +GLU 1.3 ± 0.15 mM; +GLU + MPP 2.7 ± 0.4 mM), fumarate, and a-ketoglutarate (<10 μM) while a range of other metabolic organic acids remained undetected. Amino acids quantified by o-phthalaldehyde precolumn derivatization/electrochemical detection–LC show accumulation of l-alanine (1.6 ± .052 mM), l-glutamate (285 ± 9.7 μM), l-asparagine (202 ± 2.1 μM), and l-aspartate (84.2 ± 4.9 μM) produced during routine metabolism, while other amino acids remain undetected. In contrast, the data show no evidence for accumulation of acetaldehyde, aldehydes, or ketones (Purpald/2,4-dinitrophenylhydrazine—Brady's reagent), acetoin (Voges–Proskauer test), or alcohols (NAD⁺-linked alcohol dehydrogenase). In conclusion, these results provide preliminary evidence to suggest the existence of an active pyruvate–alanine transaminase or phosphotransacetylase/acetyl-CoA synthetase pathway to be involved with anaerobic energy metabolism of cancer cells.     

γ-Glutamyl 16-diaminopropane derivative of vasoactive intestinal peptide: a potent anti-oxidative agent for human epidermoid cancer cells

We previously demonstrated that the γ-glutamyl 16 amine derivative of vasoactive intestinal peptide (VIP) acts as structural VIP agonist with affinity and potency higher than VIP. Herein, we have evaluated the effects of VIP and γ-Gln16-diaminopropane derivative of VIP (VIP-DAP3) on the proliferation and protection from oxidative stress induced by hydrogen peroxide (H₂O₂) on epidermoid carcinoma cell lines. We have found that 10⁻¹¹ M VIP-DAP3 completely antagonized the inhibition induced by H₂O₂ on both cell proliferation and S-phase distribution while these effects were only partially antagonized by equimolar concentrations of VIP. Moreover, both oxidative stress and intracellular lipid oxidation induced by H₂O₂ were reduced by VIP and completely antagonized by VIP-DAP3. Thereafter, we have found that H₂O₂ increased p38 kinase activity and both HSP70 and HSP27 expression. VIP and VIP-DAP3 again antagonized these effects partially or totally, respectively. H₂O₂ reduced the activity of extracellular signal-regulated kinases Erk-1/2 and Akt, signalling proteins involved in proliferation/survival pathways. Again VIP restored the activity of both kinases while VIP-DAP3 caused indeed an increase of their activity as compared to untreated cells. These data suggest that VIP-DAP3 has a stronger anti-oxidative activity as compared to VIP likely based on its super-agonistic binding on the putative receptor.     

Molecular dynamics simulations of nafion and sulfonated poly ether sulfone membranes II. Dynamic properties of water and hydronium

We measured the self-diffusion coefficients of water in a Nafion membrane and two sulfonated polyethersulfone (SPES) membranes with varying ion-exchange capacities (IEC) in terms of relative humidity using the pulse field gradient NMR (PFG-NMR) technique. The self-diffusion coefficients were plotted against the number of water molecules per sulfonic acid group, λ, and compare these values with the results of molecular dynamics (MD) simulations. Classical MD simulations for all membranes were carried out using a consistent force field at λ = 3, 6, 9, 12, and 15. The dynamic properties of water (H₂O) and hydronium (H₃O⁺) on a molecular level were estimated as self-diffusion coefficients and residence times around a sulfonate group ( \textSO₃^- {\text{SO}}_3^{-} ). The diffusion coefficients of H₂O and H₃O⁺ followed the order, Nafion > SPES with IEC = 1.4 > SPES with IEC = 1.0 > SPES with IEC = 0.75, which agreed with the experimental data. The residence time distribution of H₂O around \textSO₃^- {\text{SO}}_3^{-} in Nafion was in the range of 1–6 ps, whereas H₂O in the SPES exhibited a residence time of greater than 20 ps.     

Induction of alkalinization and an oxidative burst by low doses of cycloheximide in soybean cells

Soybean (Glycine max L. Merr.) Cell-suspension cultures inoculated with avirulent Pseudomonas syringae pv. glycinea bacteria generated a sustained oxidative burst 3–6 h after the infection. The H₂O₂ production was not dependent on protein biosynthesis but, surprisingly, cycloheximide itself was a very strong inducer of the oxidative burst and of the alkalinization measured in the cell culture medium. Both responses were activated in a very similar manner by inhibitors of protein phosphatases, implicating a phosphorylation change evoked by cycloheximide as a trigger for the elicitation. The activation of the oxidative burst was totally blocked by the kinase inhibitor K252a. The alkalinization response preceded the oxidative burst. The generation of H₂O₂ depleted the medium of H⁺ but the expected alkalinization of about one pH-unit did not occur. The H₂O₂ production by the plasma membrane oxidase must therefore be charge-compensated, likely via H⁺-channel activity. Received: 4 October 1997 / Accepted: 12 May 1998     

Mycotoxins in horse feed

A total of 62 samples of commercial horse feed preparations (complementary feeds) containing cereal mixtures (“muesli” or mash, n = 39; pelleted feeds, n = 12), and plain horse feed grains (maize, n = 5; oats, n = 4; barley, n = 2) were purchased from 21 different producers/distributors from the German market. All samples were analysed by competitive enzyme immunoassays (EIA) for six different mycotoxins (mycotoxin groups). Analytes (detection limit, mean recovery) were: deoxynivalenol (DON, 10 μg/kg, 84%), zearalenone (ZEA, 5 μg/kg, 93%), fumonisin B₁ (FB₁, 2 μg/kg, 113%), T-2 toxin (T-2, 0.1 μg/kg, 71%), sum of T-2 + HT-2 toxin (T-2/HT2, 0.2 μg/kg, 97%), ochratoxin A (OTA, 0.2 μg/kg, 67%), and total ergot alkaloids (Generic Ergot Alkaloids “GEA”, 30 μg/kg, 132%). All samples contained DON (16–4,900 μg/kg, median 220 μg/kg), T-2/HT-2 (0.8–230 μg/kg, median 24 μg/kg), and T-2 (0.3–91 μg/kg, median 7 μg/kg). ZEA was detected in 98% of the samples (7–310 μg/kg, median 61 μg/kg). Most samples (94%) were positive for FB₁ (2–2,200 μg/kg, median 27 μg/kg). Ergot alkaloids were detected in 61% of samples (28–1,200 μg/kg, median 97 μg/kg), OTA was found in 42% of samples (0.2–4 μg/kg, median 0.35 μg/kg). The results demonstrate that a co-contamination with several mycotoxins is very common in commercial horse feed from the German market. The toxin concentrations were in most cases well below the levels which are usually considered as critical or even toxic. The highest mycotoxin concentrations were mostly found in single-grain cereal feed: the maximum values for DON and FB₁ were found in maize, the highest T-2/HT-2 toxin concentrations were found in oats, and the highest concentration of ergot alkaloids was found in barley. In composed feeds, no correlation between cereal composition and mycotoxin levels could be found.     

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

 Reactions (25  °C) of galactose oxidase, GOase_ox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH₂OH:- D-galactose (I) and 2-deoxy-d-galactose (II) (monosaccharides); methyl-β-d-galactopyranoside (III) (glycoside);d-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O₂. The GOase_ox state has a tyrosyl radical coordinated at a square-pyramidal Cu^II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase_ox] (0.8–10 μM), RCH₂OH (1.0–6.0 mM), and O₂ (0.14–0.29 mM), with I=0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O₂. Each trace was fitted to the competing reactions GOase_ox+RCH₂ OH → GOase_redH₂+RCHO (k ₁), and GOase_redH₂+O₂→ GOase_ox+H₂O₂ (k ₂), with GOase_redH₂ written as the doubly protonated two-electron-reduced Cu^I product. It was necessary to avoid auto-redox interconversion of GOase_ox and GOase_semi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in ∼3 h. At pH >7.5, rate constants 10^–4 k ₁ / M^–1 s^–1 for the reactions of GOase_ox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k ₁ values decreased by factors of up to a half, and acid dissociation pK _as in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOase_ox gave an independently determined pK _a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pK _a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k ₂ (1.01×10⁷ M^–1 s^–1) is independent of pH in the range 5.5–9.0 investigated, suggesting that H⁺ (or H-atoms) for the O₂ → H₂O₂ change are provided by the active site of GOase_red . The Cu^I of GOase_red is less extensively complexed, and a coordination number of three is likely. Received: 4 February 1997 / Accepted: 16 May 1997     

A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV–vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0–10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H₂O₂ without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H₂O₂ in the range 5 × 10⁻⁷–3 × 10⁻⁴ M with a detection limit of 6 × 10⁻⁸ M. The response shows Michaelis–Menten behavior at higher H₂O₂ concentrations and the apparent Michaelis–Menten constant is estimated to be 112 μM.     

Permeabilization of <Emphasis Type="Italic">Microbacterium oxylans</Emphasis> shifts the conversion of puerarin from puerarin-7-<Emphasis Type="Italic">O</Emphasis>-glucoside to puerarin-7-<Emphasis Type="Italic">O</Emphasis>-fructoside

The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 ± 31.9 μM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 ± 48.0 μM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, ¹³C NMR, ¹H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 ± 5.4 μM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 ± 27.7 μM puerarin-7-O-glucoside and 187.8 ± 29.5 μM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 ± 24.0 μM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product’s composition.