The mechanism of action of organic mercury compounds on cytochrome oxidase.
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Cytochromes↗
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Cellular antioxidant effects of atorvastatin in vitro and in vivo.
3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert direct effects on vascular cells and beneficially influence endothelial dysfunction. Because reactive oxygen species (ROS) may lead to vascular damage and dysfunction, we investigated the effect of atorvastatin on ROS production and the underlying mechanisms in vitro and in vivo. Cultured rat aortic vascular smooth muscle cells were incubated with 10 micromol/L atorvastatin. Angiotensin II-induced and epidermal growth factor-induced ROS production were significantly reduced by atorvastatin (dichlorofluorescein fluorescence laser microscopy). Atorvastatin downregulated mRNA expression of the NAD(P)H oxidase subunit nox1, whereas p22phox mRNA expression was not significantly altered (reverse transcription-polymerase chain reaction, Northern analysis). Membrane translocation of rac1 GTPase, which is required for the activation of NAD(P)H oxidase, was inhibited by atorvastatin (Western blot). mRNA expression of superoxide dismutase isoforms and glutathione peroxidase was not modified by atorvastatin, whereas catalase expression was upregulated at mRNA and protein levels, resulting in an increased enzymatic activity. Effects of atorvastatin on ROS production and nox1, rac1, and catalase expression were inhibited by L-mevalonate but not by 25-hydroxycholesterol. In addition, spontaneously hypertensive rats were treated with atorvastatin for 30 days. ROS production in aortic segments was significantly reduced in statin-treated rats (lucigenin chemiluminescence). Treatment with atorvastatin reduced vascular mRNA expression of p22phox and nox1 and increased aortic catalase expression. mRNA expression of superoxide dismutases, glutathione peroxidase, and NAD(P)H oxidase subunits gp91phox, p40phox, p47phox, and p67phox remained unchanged. Translocation of rac1 from the cytosol to the cell membrane was also reduced in vivo. Thus, atorvastatin exerts cellular antioxidant effects in cultured rat vascular smooth muscle cells and in the vasculature of spontaneously hypertensive rats mediated by decreased expression of essential NAD(P)H oxidase subunits and by upregulation of catalase expression. These effects of atorvastatin may contribute to the vasoprotective effects of statins.
Animals↗
Design and characterization of immobilized enzymes in microfluidic systems.
Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.
Bacterial Proteins↗
Ferrocene-conjugated m-phenylenediamine conducting polymer-incorporated peroxidase biosensors.
The development and characteristics of a reagentless amperometric biosensor employing horseradish peroxidase incorporated in an electrochemically deposited ferrocene-modified phenylenediamine film on a glassy carbon electrode is reported. The horseradish peroxidase/poly(m-aminoanilinomethylferrocene)- modified glassy carbon electrode reagentless biosensor measured hydrogen peroxide and other organic peroxides in both aqueous and organic medium by reduction at a low applied potential of -0.05 V (vs Ag/AgCl) without interference from molecular oxygen. When modified with glucose oxidase, the new bienzyme electrode measured glucose sensitively and selectively, demonstrating the suitability of the above peroxide biosensor for other oxidoreductase enzyme-based biosensors.
Biosensing Techniques↗
Patterning enzymes inside microfluidic channels via photoattachment chemistry.
We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.
Animals↗
[Leukocyte oxidase and hemoglobin].
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Coloring Agents↗
Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.
To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.
Arachidonate 5-Lipoxygenase↗
Xanthine oxidase and liver nitrogen variation with dietary protein.
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Coloring Agents↗
Neutrophil activation after skeletal muscle ischemia in humans.
The aim of the study was to investigate the time course of neutrophil activation after skeletal muscle ischemia in humans and to assess the effect of xanthine oxidase inhibitor allopurinol or cyclooxygenase inhibitor indomethacin. In patients undergoing tourniquet ischemia of the upper limb, polymorphonuclear neutrophils (PMN) were simultaneously isolated from antecubital vein blood of both the contralateral control arm and the tourniquet arm. PMN-superoxide production (PMN-SOP) was determined by a cytochrome C reduction assay, PMN-myeloperoxidase activity (PMN-MPO) by guaiacol oxidation and serum PMN-elastase concentration by an enzyme immunoassay. At 60 min after release of the tourniquet, significant increases of PMN-SOP, PMN-MPO, and serum elastase concentrations were observed in tourniquet arms as compared with control arms (p < .05). Allopurinol (300 mg orally, 12 and 2 h before ischemia) significantly inhibited the increase of PMN-SOP, PMN-MPO, and serum elastase (p < .05). Indomethacin (50 mg orally, 2 h before ischemia) prevented increased PMN-MPO and serum elastase, but prevented increased PMN-SOP only when neutrophils were incubated in the presence of their autologous plasma. These findings suggest that ischemia/reperfusion of human skeletal muscle involves both xanthine oxidase-dependent oxygen free radicals and cyclooxygenase metabolites. These pathways could activate circulating neutrophils which potentially inflict local and remote endothelial injury.
Adolescent↗
Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance.
Tissue-specific or regulated expression of transgenes is desirable in order to prevent pleiotropic side effects of putatively harmful transgene products as well as loss of energy resources due to unnecessary accumulation of transgene products. Epidermis-specific expression would be useful for many defense-related genes directed against attack by fungal pathogens that enter the plant body by direct penetration through the epidermis. In an approach to enhance resistance of wheat to the powdery mildew fungus Blumeria graminis f.sp. tritici, a novel epidermis-specific promoter was developed and used for expression of two defense-related genes. A 2.3 kb fragment of the wheat GstA1 promoter in combination with an intron-containing part of the wheat WIR1a gene was found to drive strong and constitutive transient expression in wheat epidermis. This promoter-intron combination was used for overexpression of oxalate oxidase 9f-2.8 and TaPERO peroxidase, two defense-related wheat genes expressed in inner leaf tissues. Expression studies of several transgenic lines by in situ oxalate-oxidase staining, RNA and protein blot analyses, as well as real-time PCR, demonstrated strong and constitutive transgene expression in the shoot epidermis. Transient as well as stable over-expression of the TaPERO peroxidase gene in wheat epidermis under the control of the GstA1i promoter resulted in enhanced resistance against Blumeria graminis f.sp. tritici, whereas oxalate-oxidase overexpression had no effect in either system. The data suggest that the wheat GstA1 promoter in combination with the WIR1a intron is useful for transgenic approaches to fungal disease resistance in cereals.
Ascomycota↗
Determination of L-glutamate using flow injection analysis with immobilized L-glutamate oxidase reactor.
L-Glutamate oxidase (GOD) and horseradish peroxidase (HRP) were covalently coupled on alkylamine pretreated controlled pore glass (CPG) by means of glutaraldehyde. The immobilized enzymes were packed into a teflon tube and used in flow injection analysis (FIA) system for L-glutamate determination. A good linearity range was obtained at 0.1-2.0 mM, and the coefficient of variation was 0.7% (n = 8). More than 80 samples were measured within an hour. The stability of the immobilized GOD reactor was good, retaining 50% of its initial activity after 4 months storage in buffer at 4 degrees C. When the concentration of L-glutamate remained lower than 2.5 mM, the determination of L-glutamate in this system was not affected by pH and temperature within the range of 6.0-8.0 and 20-35 degrees C, respectively. The system was applied to determine L-glutamate in broth samples during L-glutamate fermentation and good correlations were achieved between results obtained with the FIA system, L-glutamate oxidase kit and Warburg's method.
Amino Acid Oxidoreductases↗
Abscisic acid induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings.
The changes in the activity of peroxidase (POD) extracted from the cell wall and the level of H(2)O(2) of rice seedling roots treated with abscisic acid (ABA) and their correlation with root growth were investigated. Increasing concentrations of ABA from 3 to 18 µM progressively reduce root growth and increase POD activities (using guaiacol or ferulic acid as a substrate) extracted from the cell wall of rice roots. The reduction of root growth by ABA is also correlated with an increase in H(2)O(2) level. Both diamine oxidase (DAO) and NADH peroxidase (NADH-POD) are known to be responsible for the generation of H(2)O(2). ABA treatment increased NADH-POD and DAO activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the generation of H(2)O(2) in the cell wall of ABA-treated roots. An increase in the level of H(2)O(2) and the activity of POD extracted from the cell wall of rice roots preceded root growth reduction caused by ABA. An increase in DAO and NADH-POD activities coincided with an increase in H(2)O(2) in roots caused by ABA. Since DAO catalyzes the oxidation of putrescine, the results that ABA increases the activity of DAO in roots is consistent with those that ABA decreases the level of putrescine. In conclusion, cell wall stiffening catalyzed by POD is possibly involved in the regulation of root growth reduction caused by ABA.
Journal Article↗
A reagentless optical biosensor based on the intrinsic absorption properties of peroxidase.
During the reversible reaction between peroxidase (HRP) and H(2)O(2), several peroxidase intermediate species, showing different molecular absorption spectra, are formed which can be used for H(2)O(2) determination; when H(2)O(2) is generated in a previous enzymatic reaction, the substrate involved in this reaction can also be determined. On this basis, a new family of fully reversible reagentless optical biosensors containing HRP is presented; glucose determination is used as a model. The biosensor (which can be used for at least 6 months and/or more than 750 measurements) is prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix. A mathematical model (in which optical, kinetic and transport aspects are considered) relating the measured absorbance with the substrate concentration is also presented together with a simple methodology for characterization of this kind of biosensor. Regarding the optical model, the Kubelka-Mulk theory of reflectance does not give good results and the biosensors are better described by the Rayleigh theory of polymer solutions. Under working conditions, linear response ranges from 1.5x10(-6) to 3.0x10(-4)M glucose and CV was about 4%. This biosensor has been applied for glucose determination in fruit juices and synthetic serum samples without sample pretreatment.
Absorption↗
Histochemical phenotypes of von Ebner's gland of ferret and their functional implications.
Von Ebner's gland of ferret was examined by means of light microscopy, protein, mucosubstance and enzyme histochemistry, and neurohistology. Acinar cells were replete with granules containing neutral mucosubstances and disulphides, and showed strong diffuse acid phosphatase activity and weak granular staining for peroxidase. Staining for cytochrome oxidase, succinate dehydrogenase, and NADH and NAD(P)H dehydrogenases was also seen. Basolateral plasmalemma of acinar cells showed weak, ouabain-sensitive Na+,K+-ATPase activity. Ductal cells were of a simple appearance, contained thiols and showed variable staining for acid phosphatase, dehydrogenases and cytochrome oxidase. Variable amounts of beta-glucuronidase reaction product were localized in the glandular parenchyma, being marked in atrophic areas. Prominent stellate myoepithelial cells embracing acini and also basal ductal cells were demonstrated by alkaline phosphatase. Thiamine pyrophosphatase reaction product was concentrated in blood vessels around parenchyma, with little Golgi-like staining in acinar cells. Acetylcholinesterase activity was associated with an extensive network of nerve fibres embracing parenchyma, whereas catecholamine fluorescence was not seen. The results suggest that the acini of von Ebner's gland of ferret synthesise neutral secretory glycoproteins and peroxidase. Water mobilization is inconspicuous. Lysosomal activities feature in the parenchyma, possibly a consequence of processing secretory products in acini, absorption in ducts and/or adaptation atrophy. The gland receives a rich cholinergic-type innervation, and has extensive myoepithelial and microvascular networks.
Animals↗
Lipid peroxidation and antioxidant enzyme levels of intestinal renal and muscle tissues after a 60 minutes exercise in trained mice.
To investigate the effect of blood perfusion difference on oxidant status, mice were trained by a 7-week running program. Two days after the last training session, mice were exercised for 60 minutes at the same training intensity. Changes in the concentration of thiobarbituric acid reactive substance (TBARS), as an index of lipid peroxidation, in intestine, kidney and muscle, were studied in trained mice immediately (0 h), 3 h and 24 h after the running exercise and in unexercised control group. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and xanthine oxidase (XO) were determined in these tissues. Tissue SOD activities were unaffected by the exercise. Muscle GPx activity increased after exercise (0 h and 3 h group, P < 0.01) and returned to control levels at 24 h, but there was not any significant difference in intestinal and renal tissues. Renal tissue XO activity could not be determined. There was not any significant difference among groups in intestinal tissue XO activity. The activity of XO was decreased only in skeletal muscle at 0 h (P < 0.05). TBARS levels of exercised groups were higher than control in muscle (P < 0.01). Intestinal TBARS levels decreased at 0 h (P < 0.05), than reached to control level. Renal TBARS levels of 0 h and 24 h group was higher than control (P < 0.01, P < 0.01 respectively). The results show that a long distance running exercise may cause lipid peroxidation damage in skeletal muscle and kidney.
Animals↗
Replacement of the invariant lysine 77 by arginine in yeast iso-1-cytochrome c results in enhanced and normal activities in vitro and in vivo.
Oligonucleotide-directed mutagenesis of the yeast Saccharomyces cerevisiae was used to generate an abnormal iso-1-cytochrome c having an Arg-77 replacement of the normal Lys-77; this Lys-77 residue is evolutionarily conserved in most eukaryotic cytochromes c and is trimethylated in fungal and plant cytochromes c. Examination of strains having a single chromosomal copy of the gene encoding the Arg-77 protein indicated that the altered protein was synthesized at the normal rate and that it had normal or near normal activity in vivo. Examination of enzymatic activities in vitro with cytochrome b2, cytochrome c peroxidase, and cytochrome c oxidase indicated that the altered iso-1-cytochrome c has equal or enhanced catalytic efficiencies. Thus, replacement of the evolutionarily conserved residue Lys-77 produces no or only minor effects both in vivo and in vitro.
Amino Acid Sequence↗
Demonstration of monoamine oxidase type B in serotonergic and type A in noradrenergic neurons in the cat dorsal pontine tegmentum by an improved histochemical technique.
Application of a diaminobenzidine-coupled peroxidation method to monoamine oxidase (MAO) histochemistry demonstrates a detailed morphology of MAO-containing perikarya, dendrites, axons and fibers in the dorsal pontine tegmentum of the cat, sectioned with a vibratome. Further, utilization of specific inhibitors, deprenyl and clorgyline, also provides evidence that serotonergic neurons contain exclusively MAO-B and catecholaminergic ones contain MAO-A.
Animals↗
Inhibition of the lignin peroxidase of Phanerochaete chrysosporium by hydroxylamino-dinitrotoluene, an early intermediate in the degradation of 2,4,6-trinitrotoluene.
The ability of the white rot fungus Phanerochaete chrysosporium to mineralize 2,4,6-trinitrotoluene (TNT) was studied in the concentration range of 0.36 to 20.36 mg/liter. The initial rate of 14CO2 formation was 30% in 4 days at 0.36 mg of [14C]TNT per liter and decreased to 5% in 4 days at 20.36 mg of [14C]TNT per liter. Such a pronounced inhibition was not observed when a mixture of [14C]2-amino-4,6-dinitrotoluene and [14C]4-amino-2,6-dinitrotoluene was used as a substrate. 2-Hydroxylamino-4,6-dinitrotoluene and its isomer 4-hydroxylamino-2,6-dinitrotoluene were identified as the first detectable degradation products of TNT. Their transient accumulation correlated with the inhibition of TNT degradation and of the veratryl alcohol oxidase activity of lignin peroxidase. With purified lignin peroxidase H8, it could be shown that the two isomers of hydroxylamino-dinitrotoluene were oxidized by lignin peroxidase. The corresponding nitroso-dinitrotoluenes apparently were formed, as indicated by the formation of azoxy-tetranitrotoluenes.
Alcohol Oxidoreductases↗