Supplementation of alpha-tocopherol improves cardiovascular risk factors via the insulin signalling pathway and reduction of mitochondrial reactive oxygen species in type II diabetic rats

This study determined the effects of alpha- and gamma-tocopherol supplementation on metabolic control and oxidative stress in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Blood glucose, haemoglobin A1c (HbA1c), urinary protein, plasma free fatty acid, triacylglycerol and plasminogen activator inhibitor-1 (PAI-1) levels in OLETF rats were significantly higher than in non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats. Alpha-tocopherol inhibited the increase in urinary protein, blood glucose, HbA1c and PAI-1 levels, but gamma-tocopherol did not. Plasma and hepatic lipid peroxidation and hepatic steatosis were increased in OLETF rats. alpha-Tocopherol decreased lipid peroxidation. Mitochondrial reactive oxygen species production and uncoupling protein 2 (UCP2) expression were significantly increased in the heart and aorta of OLETF rats compared with LETO rats. Endothelial NO synthase and aortic nitrotyrosine were increased in OLETF rats. In contrast, the expression of phosphorylated vasodilator-stimulated phosphoprotein and glucose transporter 4 in the aorta was significantly decreased in OLETF rats. These abnormalities were reversed by alpha-tocopherol. These findings suggest that alpha-tocopherol may prevent cardiovascular tissues from oxidative stress and insulin signalling disorder resulting from diabetes mellitus.     

Gas Phase Oxidants of Cigarette Smoke Increase Nitric Oxide Synthase and Xanthine Oxidase Activities of Rabbit Brain Synaptosomes

In the present study we demonstrated that NO synthase and xanthine oxidase of synaptosomes isolated from rabbit brain cortex can be activated by the gas phase of cigarette smoke to produce nitric oxide and superoxide which react together to form peroxynitrite. Expose of synaptosomes, up to 3 hours, in the gas phase of cigarette smoke, a gradual increase in both nitric oxide and superoxide release that were inhibited by N-monomethyl-L-arginine (100 M) and oxypurinol (1 mM), respectively, was observed. NO synthase and xanthine oxidase activities were increased approximately three fold after treatment of synaptosomes with the gas phase of cigarette smoke as compared with the gas phase deprived of oxidants. Synaptosomes treated with the gas phase of cigarette smoke dramatically increased 3-nitrotyrosine production (used as an index of peroxynitrite formation). Synaptosomes treated with the gas phase of cigarette smoke, promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1,4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1,3,5-triene. Gas phase deprived of oxidants showed a small but not statistically significant (p > 0.05) effect on both malondialdehyde and membrane fluidity. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells by oxidants contained in the gas phase of cigarette smoke lead to the formation of peroxynitrite a causative factor in neurotoxicity.     

Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.     

Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: Role of hydrogen peroxide

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2·–/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 M) or histamine (100 M). Superoxide dismutase (50 U/ml), which dismutates O2·minus; into H2O2 al ient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 M were used. Buffering trace amounts of iron (o-phenanthroline; 200 M) in order to inhibit úOH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca2+-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca2+-ATPases of the endoplasmatic reticulum with thapsigargin (1 M) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 M) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O· ndash; or · OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological oxidative stress associated with a progressive increase in [Ca2+]i.     

Epr detection of endogenous nitric oxide in postischemic heart using lipid and aqueous-soluble dithiocarbamate-iron complexes

Spin-trapping techniques combined with electron paramagnetic resonance (EPR) spectroscopy to measure nitric oxide (·NO) production were compared in the ischemic-reperfused myocardium for the first time, using both aqueous-soluble and lipophilic complexes of reduced iron (Fe) with dithiocarbamate derivatives. The aqueous-soluble complex of Fe and N-methyl-D-glucamine dithiocarbamate (MGD) formed MGD2-Fe-NO complex with a characteristic triplet EPR signal (a_N12.5 G and g_iso = 2.04) at room temperature, in native isolated rat hearts following 40 min global ischemia and 15 min reperfusion. Diethyldithiocarbamate (DETC) and Fe formed in ischemic-reperfused myocardium the lipophilic DETC2-Fe-NO complex exhibiting an EPR signal (g = 2.04 and g = 2.02 at 77K) with a triplet hyperfine structure at g. Dithiocarbamate-Fe-NO complexes detected by both trapping agents were abolished by the ·NO synthase inhibitor, N^G-nitro-L-arginine methyl ester. Quantitatively, both trapping procedures provi ded similar values for tissue ·NO production, which were observed primarily during ischemia. Postischemic hemodynamic recovery of the heart was not affected by the trapping procedure. (Mol Cell Biochem 175: 91–97, 1997)     

Continuous chemotrophic growth and respiration of Chromatiaceae species at low oxygen concentrations

Endogenous and maximum respiration rates of nine purple sulfur bacterial strains were determined. Endogenous rates were below 10 nmol O₂ · (mg protein · min)^-1 for sulfur-free cells and 15–35 nmol O₂ · (mg protein · min)^-1 for cells containg intracellular sulfur globules. With sulfide as electron-donating substrate respiration rates were considerably higher than with thiosulfate. Maximum respiration rates of Thiocystis violacea 2711 and Thiorhodovibrio winogradskyi SSP1 (254.8 and 264.2 nmol O₂ · (mg protein · min)^-1, respectively) are similar to those of aerobic bacteria. Biphasic respiration curves were obtained for sulfur-free cells of Thiocystis violacea 2711 and Chromatium vinosum 2811. In Thiocystis violacea the rapid and incomplete oxidation of thiosulfate was five times faster than the oxidation of stored sulfur. A high affinity of the respiratoty system for oxygen (K _m =0.3–0.9 M O₂, V _max=260 nmol O₂ · (mg protein · min)^-1 with sulfide as substrate, K _m =0.6–2.4 M O₂, V _max=14–40 nmol O₂ · (mg protein · min)^-1 with thiosulfate as substrate), for sulfide (K _m =0.47 M, V _max=650 nmol H₂S · (mg protein × min)^-1, and for thiosulfate (K _m =5–6 M, V _max =24–72 nmol S₂O ₃ ^2- · (mg protein · min)^-1 was obtained for different strains. Respiration of Thiocystis violacea was inhibited by very low concentrations of NaCN (K _i =1.7 M) while CO concentrations of up to 300 M were not inhibitory. The capacity for chemotrophic growth of six species was studied in continuous culture at oxygen concentrations of 11 to 67 M. Thiocystis violacea 2711, Amoebobacter roseus 6611, Thiocapsa roseopersicina 6311 and Thiorhodovibrio winogradskyi SSP1 were able to grow chemotrophically with thiosulfate/acetate or sulfide/acetate. Chromatium vinosum 2811 and Amoebobacter purpureus ML1 failed to grow under these conditions. During shift from phototrophic to chemotrophic conditions intracellular sulfur and carbohydrate accumulated transiently inside the cells. During chemotrophic growth bacteriochlorophyll a was below the detection limit.     

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment ^1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded ². This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances ^{4, 5}. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation ^{4, 6}. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) ^7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators ^10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al⁷. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere ^{14, 15}. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. ^{16, 17}. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin ¹⁸. This approach proved to be sufficiently sensitive and reproducible. Download video file.^{(77M, mov)}     

Exercise conditioning attenuates the hypertensive effects of nitric oxide synthase inhibitor in rat

Many individuals with cardiovascular diseases undergo periodic exercise conditioning with or with out medication. Therefore, this study investigated the interaction of exercise training and chronic nitric oxide synthase (NOS) inhibitor (Nitro-L-Arginine Methyl Ester, L-NAME) treatment on blood pressure and its correlation with aortic nitric oxide (NO), antioxidant defense system and oxidative stress parameters in rats. Fisher 344 rats were divided into four groups: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) L-NAME (10 mg/kg, subcutaneous for 8 weeks) and (4) ET + L-NAME. Blood pressure (BP) was monitored weekly for 8 weeks with tail-cuff method. The animals were sacrificed 24 h after last treatments and thoracic aortic rings were isolated and analyzed. Exercise conditioning resulted in a significant increase in respiratory exchange ratio (RER), aortic NO production, NO synthase activity and inducible iNOS protein expression. Training significantly enhanced aortic GSH levels, GSH/GSSG ratio and up-regulation of aortic CuZn-SOD, Mn-SOD, catalase (CAT) glutathione peroxidase (GSH-Px) activity and protein expression and significantly decreased aortic lipid peroxidation. Chronic L-NAME administration resulted in a significant depletion of aortic NO, NOS activity, endothelial (eNOS) and iNOS protein expression, GSH level, GSH/GSSG ratio, down-regulation of aortic antioxidant enzyme activities and protein expressions. Aortic xanthine oxidase (XO) activity significantly increased with increased lipid peroxidation and protein oxidation after L-NAME administration. The biochemical changes were accompanied by increased in BP. Interaction of training and chronic NOS inhibitor treatment resulted in normalization of BP and aortic antioxidant enzyme activity and protein expression, up-regulation of aortic GSH/GSSG ratio, NO levels, Mn-SOD protein expression, depletion of GSSG, protein oxidation and lipid peroxidation. The data suggest that training attenuated the oxidative injury caused by chronic NOS inhibitor treatment by up-regulating the NO and antioxidant systems and lowering the BP in rats.     

Ozone detoxification in the apoplasm and symplasm of spinach,broad bean and beech leaves at ambient and elevated concentrations of ozone in air

Spinach (Spinacia oleracea L.), broad bean (Vicia faba L.) and beech (Fagus sylvatica L.) plants were exposed to ozone at concentrations often measured in air during the summer months (120–300 g·m^–3) and antioxidants were determined in the leaf tissue and in the aqueous phase of the cell wall, the apoplasm. Concentrations of both reduced ascorbate (AA) and its oxidized form, dehydroascorbate (DHA), showed the tendency to increase transiently in the apoplasm of spinach leaves 6–24 h after starting fumigation with ozone. In beech leaves, apoplasmic AA and DHA increased 3–7 d after beginning of treatment. At the very high concentration of 1600 g O₃·m^–3, an increase of apoplasmic AA was already measured after 1 d in beech leaves. Apparently, spinach and beech leaves respond to oxidative stress by increasing AA transport into the apoplasm and by accelerating DHA export. In contrast to these observations, DHA accumulated during 3 d of fumigation with only 120 g O₃·m^–3 in the apoplasm of broad bean leaves, while AA contents did not increase. After termination of fumigation, the extracellular redox state of ascorbate normalized within 1 d. Glutathione could not be detected in the apoplasm of any of the three leaf species. Intracellular AA changed its redox state in response to exposure to elevated concentrations of ozone. After 4–6 weeks of fumigation with 200–300 g O₃·m^–3 an increase of intracellular DHA was measured in beech leaves. At the same time, chlorophyll contents decreased and characteristic symptoms of ozone damage could be observed. However, no significant change in the redox state of apoplasmic ascorbate could be detected in beech leaves. Evidently, detoxification of ozone by apoplasmic AA was insufficient to protect the leaf tissue. Fumigation with a high ozone concentration (1600 g·m^–3) caused an appreciable increase in the cellular contents of the oxidized forms of ascorbate and glutathione in beech leaves. Whereas in spinach leaves intracellular antioxidant contents and redox states were not altered during fumigation with 120–240 g O₃·m^–3, in broad bean leaves the intracellular DHA concentration increased and intracellular ascorbate became more oxidized after fumigation of the plants with 120 g O₃·m^–3. Apparently, broad bean leaves are more sensitive to ozone than beech and spinach leaves.Abbreviations AA ascorbate, reduced form - DHA ascorbate, oxidized form (dehydroascorbate) - FW fresh weight - GSH glutathione, reduced form - GSSG glutathione, oxidized form - IWF intercellular washing fluid - V_air intercellular air space volume of leaves - V_apo apoplasmic water volume of leaves This work was supported within the Sonderforschungsbereich 251 of the University of Würzburg.     

A continuous culture study of the phosphorus nutrition of Rhizobium trifollii WU95, Rhizobium NGR234 and Bradyrhizobium CB756

With continuous cultures in a fully defined minimal salts medium steady states were achieved at both limiting and non-limiting concentrations of phosphate in the inflowing medium for Rhizobium trifolii WU95, cowpea Rhizobium NGR234, and Bradyrhizobium CB756.Millimolar growth yields obtained from P-limited cultures varied over 2-fold from 3.2 g dry weight·(mmol P)^-1 for WU95 to 5.3 g dry weight·(mmol P)^-1 for CB756 and 7.2 g dry weight·(mmol P)^-1 for NGR234.For both WU95 and NGR234 growth under P-excess conditions resulted in elevated levels of total biomass P and the storage compound polyphosphate, compared with P-limited cultures. However, P-limited cultures of these two strains still contained significant quantities of polyphosphate. The P-status for CB756 cultures did not affect either total biomass P or polyphosphate levels. Alkaline phosphatase was maximally derepressed in P-limited cultures of WU95 and NGR234. However, in CB756 alkaline phosphatase was not detected at significant levels regardless of its P supply.These data suggest that growth of rhizobia is controlled predominantly by the attainment of a critical internal P level.Abbreviation HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid     

Activities of some oxidative and hydrolytic enzymes in musculus biceps brachii of rats after tonic stress

Summary Employing histochemical and biochemical techniques the authors studied the activities of some oxidative and hydrolytic enzymes in musculus biceps brachii of rats which from day 15 of life (at this time the muscle is differentiated) were stressed by tonic exercise on ladders. After six months of stress significant changes in the proportions of basic fiber types were observed (an increase of intermediate and decrease of white fibers). Significant changes were found in the activities of succinate dehydrogenase, -glucan phosphorylase, and nonspecific esterase (increase of activities), whereas no such differences were found in the activities of lactate dehydrogenase and mitochondrial -glycerol phosphate dehydrogenase. The difficulties encountered in the classification of basic fiber types are pointed out. The findings furnished evidence that tonic stress influences the metabolism and typology of the striated muscle of the growing rat.     

Changes in carp myosin ATPase induced by temperature acclimation

Summary Myosins were isolated from dorsal ordinary muscles of carp acclimated to 10°C and 30°C for a minimum of 5 weeks and examined for their ATPase activities. Ca²⁺-ATPase activity was different between myosins from cold-and warm-acclimated carp, especially at KCl concentrations ranging from 0.1 to 0.2 M, when measured at pH 7.0. The highest activity was 0.32 mol P_i·min^-1·mg^-1 at 0.2 M KCl for cold-acclimated carp and 0.47 mol P_i·min^-1·mg^-1 at 0.1 M KCl for warm-acclimated fish. The pH-dependency of Ca²⁺-ATPase activity at 0.5 M KCl for both carp was, however, similar exhibiting two maxima around 0.3 mol P_i·min^-1·mg^-1 at pH 6 and 0.4 mol P_i·min^-1·mg^-1 at pH 9. K⁺(EDTA)-ATPase activity at pH 7.0 neither exhibited differences between both myosins. It increased with increasing KCl concentration showing the highest value of about 0.4 mol P_i·min^-1·mg^-1 at 0.6–0.7 M KCl. Actin-activated myosin Mg²⁺-ATPase activity was markedly different between cold-and warm-acclimated carp. The maximum initial velocity was 0.53 mol P_i·min^-1·mg^-1 myosin at pH 7.0 and 0.05 M KCl for cold-acclimated carp, which was 1.6 times as high as that for warm-acclimated carp. These differences were in good agreement with those obtained with myofibrillar Mg²⁺-ATPase activity between both carp. No differences were, however, observed in myosin affinity to actin. Differences in myosin properties between cold- and warm-acclimated carp were further evidenced by its thermal stability. The inactivation rate constant of myosin Ca²⁺-ATPase was 25·10^-4·s^-1 at 30°C and pH 7.0 for cold-acclimated carp, which was about 4 times as high as that for warm-acclimated carp. Light chain composition did not differ between both carp myosins. The differences in a primary structure of the heavy chain subunit was, however, clearly demonstrated between both myosins by peptide mapping.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5 dithio-bis-2-nitrobenzoic acid - DTT dithiothreitol - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Respiration rates of two species of Gnathostomulids

Summary Respiration rates for two species of Gnathostomulida from poorly oxygenated subtidal sands of Bermuda were measured using Cartesian diver respirometers.ForHaplognathia cf.ruberrima a respiration-body weight regression gaveR=0.790·W ^0,649 (in l·10^-3O₂/h and g wet weight). Respiration rates for adult animals ofGnathostomula sp. of a mean weight of 1.3 g ranged between 0.25 and 0.63 l·10^-3 O₂/h. These rates are low when compared with literature data on meiobenthic species from a wider habitat range but similar to respiration rates of marine and limnic nematodes living in sediments with strongly reducing capacity.     

Spin trapping of vascular nitric oxide using colloid Fe(II)-diethyldithiocarbamate

Currently available EPR spin-trapping techniques are not sensitive enough for quantification of basal vascular nitric oxide (NO) production from isolated vessels. Here we demonstrate that this goal can be achieved by the use of colloid Fe(DETC)(2). Rabbit aortic or venous strips incubated with 250 microM colloid Fe(DETC)(2) exhibited a linear increase in tissue-associated NO-Fe(DETC)(2) EPR signal during 1 h. Removal of endothelium or addition of 3 mM N(G)-nitro-l-arginine methyl ester (L-NAME) inhibited the signal. The basal NO production was estimated as 5.9 +/- 0.5 and 8.3 +/- 2.1 pmol/min/cm(2) in thoracic aorta and vena cava, respectively. Adding sodium nitrite (10 microM) or xanthine/xanthine oxidase in the incubation medium did not modify the intensity of the basal NO-Fe(DETC)(2) EPR signal. Reducing agents were not required with this method and superoxide dismutase activity was unchanged by the Fe(DETC)(2) complex. We conclude that colloid Fe(DETC)(2) may be a useful tool for direct detection of low amounts of NO in vascular tissue.     

Antioxidant depletion in aortic crossclamping ischemia: increase of the plasma alpha-tocopheryl quinone/alpha-tocopherol ratio.

To assess whether oxidative stress contributes to the ischemia/reperfusion injury of aortic surgery, the contents of alpha-tocopherol, alpha-tocopheryl quinone, ascorbate, lipid-derived malondialdehyde, protein thiols, cholesterol, and lactate were analyzed in plasma samples from 24 patients subjected to aortic crossclamping. alpha-Tocopherol, ascorbate, and protein thiols decreased during ischemia, whereas alpha-tocopheryl quinone increased in all but two cases, doubling on average in proportion to alpha-tocopherol. Upon reperfusion alpha-tocopherol, ascorbate, and protein thiols remained low, whereas alpha-tocopheryl quinone returned to the preischemic level. Lipid-derived malondialdehyde (a measure of lipid hydroperoxides) increased significantly only during reperfusion. The results suggest that oxidative stress occurs simultaneously with ischemia/reperfusion during aortic surgery, and that measurement of the tocopheryl quinone/tocopherol ratio may shed new light on the underlying pathological events.     

Nitric Oxide Synthase Inhibition Prevents Acute Quinolinate-Induced Striatal Neurotoxicity

Quinolinic acid (QUIN) is an endogenous excitotoxin acting on N-methyl-D-aspartate (NMDA) receptors, that leads to neurotoxic damage resembling the alterations observed in Huntington's disease. Two major end-points of QUIN induced neurotoxicity are both circling behavior (CB) and lipid peroxidation (LP). Recently, nitric oxide (NO) has been implicated as a mediator of cell injury in some neurological disorders, thus, NO as a free radical might be involved in QUIN-induced neurotoxicity and oxidative stress. In the present study we evaluated the possible role of NO on QUIN-induced neurotoxicity, by measuring nitric oxide synthase activity (NOS), before and after QUIN-induced damage and by evaluating the effect of NOS inhibition on acute QUIN-induced CB and LP. Rats were striatally microinjected with QUIN (240 nmol/1l). QUIN administration increased NOS activity by 327% as compared to control values and this enhancement was inhibited by i.v. pretreatment with a NOS inhibitor the N^G-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg). QUIN-induced CB was also attenuated by pretreatment of rats with 1, 5, 10 and 15 mg/kg of L-NAME by –37, –55, –62 and –74% vs QUIN respectively. Similarly, L-NAME also reduced by 32% the QUIN-induced LP. These findings suggest that enhanced NOS activity may participate in QUIN-induced neurotoxicity and oxidative stress.     

Effects of CO2 enrichment and water stress on gas exchange of Liquidambar styraciflua and Pinus taeda seedlings grown under different irradiance levels

Summary The effects of CO₂ enrichment and water stress on gas exchange of Liquidambar styraciflua L. (sweetgum) and Pinus taeda L. (loblolly pine) seedlings were examined for individuals grown from seed under high (1000 mol·m^-2·s^-1) and low (250 mol·m^-2·s^-1) photosynthetic photon flux density at 350, 675 and 1000 l·l^-1 CO₂. At 8 weeks of age, half the seedlings in each CO₂-irradiance treatment were subjected to a drying cycle which reduced plant water potential to about -2.5 MPa in the most stressed plants, while control plants remained well-watered (water potentials of -0.3 and -0.7 MPa for sweetgum and loblolly pine, respectively). During this stress cycle, whole seedling net photosynthesis, transpiration and stomatal conductance of plants from each CO₂-irradiance-water treatment were measured under respective growth conditions.For both species, water stress effects on gas exchange were greatest under high irradiance conditions. Waterstressed plants had significantly lower photosynthesis rates than well-watered controls throughout most of the drying cycle, with the most severe inhibition occurring for low CO₂, high irradiance-grown sweetgum seedlings. Carbon dioxide enrichment had little effect on gas exchange rates of either water-stressed or well-watered loblolly pine seedlings. In contrast, water stress effects were delayed for sweetgum seedlings grown at elevated CO₂, particularly in the 1000 l·l^-1 CO₂, high irradiance treatment where net photosynthesis, transpiration and conductance of stressed plants were 60, 36 and 33% of respective control values at the end of the drying cycle. Development of internal plant water deficits was slower for stressed sweetgum seedlings grown at elevated CO₂. As a result, these seedlings maintained higher photosynthetic rates over the drying cycle than stressed sweetgum seedlings grown at 350 l·l^-1 CO₂ and stressed loblolly pine seedlings grown at ambient and enriched CO₂ levels. In addition, water-stressed sweetgum seedlings grown at elevated CO₂ exhibited a substantial increase in water use efficiency.The results suggest that with the future increase in atmospheric CO₂ concentration, sweetgum seedlings should tolerate longer exposure to low soil moisture, resulting in greater first year survival of seedlings on drier sites of abandoned fields in the North Carolina piedmont.     

Inhibitory effect of curcumin on the invasion of rat ascites hepatoma cells in vitro and ex vivo

Curcumin, a yellow pigment in turmeric, is a food factor withantioxidative activity. The effect of curcumin on the proliferation and invasion of the rat ascites hepatoma AH109Acells was studied in vitro and ex vivo assay systems. Especially, a co-culture system of the hepatoma cellswith mesothelial cells derived from rat mesentery was employed to investigate the invasive motility. Curcumin suppressed thehepatoma slipping motility in a dose-dependent manner up to 5 M and thereafter maintained the effect up to 20 M, whereas this substance exerted little influence on the proliferation of the hepatoma cells at the same concentrations. Sera obtained from rats orally given curcumin also inhibited the AH109A cellular invasive movement when added to the culturemedium. Hepatoma cells previously cultured with hypoxanthineand xanthine oxidase showed a highly invasive activity. Curcumin and curcumin-loaded rat sera suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with hypoxanthine, xanthine oxidase and either of curcumin samples. These resultssuggest that the antioxidative property of curcumin may beinvolved in its anti-invasive action.     

Identification of xanthine and hypoxanthine as components of assembly pheromone in excreta of argasid ticks

In addition to guanine, xanthine and hypoxanthine were identified in white spherules in excreta of five species ofArgas andOrnithodoros ticks by a reverse-phase high-performance liquid chromatography (HPLC) and a gas chromatographic method with mass spectrometric detection, (GC/MS). The mutual relationships of these purines in excreta ofArgas (Persicargas) persicus were found to be less than 1.5% for hypoxanthine, less than 9.0% for xanthine and 89.8–98.6%, for guanine. In excreta of other species, the relationships of purines were similar, with the exception ofArgas (A.) reflexus andA. (A.) polonicus, where the amount of hypoxanthine was rather elevated. Uric acid was also identified in some cases. The assembly efficacy of xanthine and hypoxanthine is similar to that of guanine, but xanthine significantly enhances the assembly efficacy of commercial guanine when mixed in ratio of about 125. Thus, xanthine seems to be the second important component of assembly pheromone of argasid ticks.     

Nitrate induction in spruce: an approach using compartmental analysis

Using ¹³NO ₃ ^– -efflux analysis, the induction of nitrate uptake by externally supplied nitrate was monitored in roots of intact Picea glauca (Moench) Voss. seedlings over a 5-d period. In agreement with our earlier studies, efflux analysis revealed three compartments, which have been identified as surface adsorption, apparent free space, and cytoplasm. While induction of nitrate uptake was pronounced, NO ₃ ^– fluxes in induced plants were decidedly lower and the induction response was slower than in other species. Influx rose from 0.1 mol·g^–1·h^–1 (measured at 100 M [NO ₃ ^– _o) in uninduced plants to a maximum of 0.5 mol·g^–1h^–1 after 3 d of exposure to 100 M [NO ₃ ^– _o and declined to 0.3–0.4 mol·g^–1h^–1 at the end of the 5-d period. Efflux remained relatively constant around 0.02-0.04 mol·g^–1h^–1, but its percentage with respect to influx declined from initially high values (around 30%) to steady-state values of 4–7%. Cytoplasmic [NO ₃ ^– ] ranged from the low micromolar in uninduced plants to a maximum of 2 mM in plants fully induced at 100 M [NO ₃ ^– ]_o. In-vivo root nitrate reductase activity (NRA) was measured over the same time period, and was found to follow a similar pattern of induction as influx. The maximum response in NRA slightly preceded that of influx. It increased from 25 nmol·g^–1·h^–1 without prior exposure to NO ₃ ^– to peak values around 150 nmol· g^–1h^–1 after 2 d of exposure to 100 M [NO ₃ ^– ]_o. Subsequently, NRA declined by about 50%. The dynamics of flux partitioning to reduction, to the vacuole, the xylem, and to efflux during the induction process are discussed.The research was supported by an Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. J. Vidmar for essential assistance in experiments.