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The combined effect of drought stress and heat shock on gene expression in tobacco.

In nature, plants encounter a combination of environmental conditions that may include stresses such as drought or heat shock. Although drought and heat shock have been extensively studied, little is known about how their combination affect plants. We used cDNA arrays, coupled with physiological measurements, to study the effect of drought and heat shock on tobacco (Nicotiana tabacum) plants. A combination of drought and heat shock resulted in the closure of stomata, suppression of photosynthesis, enhancement of respiration, and increased leaf temperature. Some transcripts induced during drought, e.g. those encoding dehydrin, catalase, and glycolate oxidase, and some transcripts induced during heat shock, e.g. thioredoxin peroxidase, and ascorbate peroxidase, were suppressed during a combination of drought and heat shock. In contrast, the expression of other transcripts, including alternative oxidase, glutathione peroxidase, phenylalanine ammonia lyase, pathogenesis-related proteins, a WRKY transcription factor, and an ethylene response transcriptional co-activator, was specifically induced during a combination of drought and heat shock. Photosynthetic genes were suppressed, whereas transcripts encoding some glycolysis and pentose phosphate pathway enzymes were induced, suggesting the utilization of sugars through these pathways during stress. Our results demonstrate that the response of plants to a combination of drought and heat shock, similar to the conditions in many natural environments, is different from the response of plants to each of these stresses applied individually, as typically tested in the laboratory. This response was also different from the response of plants to other stresses such as cold, salt, or pathogen attack. Therefore, improving stress tolerance of plants and crops may require a reevaluation, taking into account the effect of multiple stresses on plant metabolism and defense.

Adaptation, Physiological↗

Indole-3-acetic Acid Oxidase from Peas: I. Occurrence and Distribution of Peroxidative and Nonperoxidative Forms.

Tissues of etiolated pea seedlings variety Alaska were examined for the presence of peroxidative and nonperoxidative forms of indoleacetic acid (IAA) oxidase. Enzymes were extracted in a sequence involving acetone powder preparation from pea tissues, buffer extraction of the powder, ammonium sulfate precipitation, dialysis, lyophilization, and acrylamide gel electrophoresis. Electrophoretically separable proteins were assayed for IAA oxidase activity with the Salkowski test, and peroxidase activity was based on the color reaction with benzidine and H(2)O(2). Each tissue examined contained several nonperoxidative IAA oxidases. No tissue contained more than three peroxidative IAA oxidases, whereas the plumule hooks (a tissue with a high IAA oxidase activity) contained no detectable peroxidases. The results indicate that nonperoxidative IAA oxidases might play a major role in the regulation of IAA content in pea seedlings.

Journal Article↗

IgA is a more potent inducer of NADPH oxidase activation and degranulation in blood eosinophils than IgE.

Human eosinophils can mediate both beneficial and detrimental responses in parasitic and allergic diseases. Binding of aggregated immunoglobulin to Fc receptors on eosinophils mediates important defence processes, including generation of activated oxygen species resulting from NADPH oxidase activation, and eosinophil peroxidase release following degranulation. The abilities of a matched set of IgA, IgG and IgE antibodies to elicit such responses in blood-derived eosinophils were compared using a chemiluminescence assay. IgA and IgG, but not IgE, were found to trigger NADPH oxidase activation and degranulation in eosinophils. This non-responsiveness to IgE did not result from receptor blockade by endogenous IgE since no blood-derived IgE was detectable on freshly isolated eosinophils. Moreover, while cross-linking of FcalphaRI by specific mAbs triggered NADPH oxidase activation and degranulation in blood-derived eosinophils, equivalent cross-linking of FcvarepsilonRI or FcvarepsilonRII did not elicit such responses. Therefore IgA is more potent at eliciting activated oxygen species release and degranulation in eosinophils than IgE, suggesting that the importance of IgA in eosinophil activation in immune defence and allergy may have been underestimated.

Cell Degranulation↗

Some properties of the H2O2/O2- generating system from the lignifying xylem of Zinnia elegans.

The properties of the enzymatic system responsible for generating H2O2/O2- in the lignifying xylem of Zinnia elegans were studied using the starch/KI method for monitoring H2O2 production and the nitroblue tetrazolium method for monitoring superoxide anion production. The results showed that H2O2/O2- production by lignifying xylem tissues was insensitive to inhibitors of peroxidase and poly(di)amine oxidases. However, H2O2/O2 production in the xylem of Z. elegans was sensitive to the inhibitors of phagocytic plasma membrane NADPH oxidase, pyridine, imidazole, quinacrine and diphenylene iodonium. The sensitivity of H2O2/O2- production to the respective inhibitors of calmodulin (R-24571), phospholipase C (neomycin sulfate), and protein kinase (staurosporine), and its reversion by an inhibitor of protein phosphatases (cantharidin); pointed to the analogies existing between the mechanism of H2O2/O2- production in the lignifying xylem of Z. elegans and the oxidative burst observed during the hypersensitive plant cell response. These results suggest the existence of a metabolic cascade involving calmodulin, IP3 and protein phosphorylation in the activation of the enzymatic system responsible for H2O2/O2- production in the lignifying xylem of Z. elegans.

Asteraceae↗

Phenylethylamine-induced generation of reactive oxygen species and ascorbate free radicals in tobacco suspension culture: mechanism for oxidative burst mediating Ca2+ influx.

In the previous paper [Kawano et al. (2000a) Plant Cell Physiol. 41: 1251], we demonstrated that addition of phenylethylamine (PEA) and benzylamine can induce an immediate and transient burst of active oxygen species (AOS) in tobacco suspension culture. Detected AOS include H2O2, superoxide anion and hydroxyl radicals. Use of several inhibitors suggested the presence of monoamine oxidase-like H2O2-generating activity in the cellular soluble fraction. It was also suggested that peroxidase(s) or copper amine oxidase(s) are involved in the extracellular superoxide production as a consequence of H2O2 production. Since more than 85% of the PEA-dependent AOS generating activity was localized in the extracellular space (extracellular fluid + cell wall), extracellularly secreted enzymes, probably peroxidases, may largely contribute to the oxidative burst induced by PEA. The PEA-induced AOS generation was also observed in the horseradish peroxidase (HRP) reaction mixture, supporting the hypothesis that peroxidases catalyze the oxidation of PEA leading to AOS generation. In addition to AOS production, we observed that PEA induced an increase in monodehydroascorbate radicals (MDA) in the cell suspension culture and in HRP reaction mixture using electron spin resonance spectroscopy and the newly invented MDA reductase-coupled method. Here we report that MDA production is an indicator of peroxidase-mediated generation of PEA radical species in tobacco suspension culture.

Amines↗

Chemiluminescent and bioluminescent techniques.

Light-emitting chemical reactions (chemiluminescence, CL) and biological reactions (bioluminescence, BL) have a diverse range of analytical applications but relatively few have been adopted by routine clinical laboratories. Advantages of CL and BL assays include sensitivity (attomole and sub-attomole detection limits), speed (signal generated in a few seconds and in some cases stable for several hours), nonhazardous reagents, and simple procedures. The most promising clinical applications are in immunoassay, protein blotting, and DNA probe assays. Chemiluminescent molecules exploited as labels include luminol, isoluminol, acridinium esters, thioesters and sulfonamides, and phenanthridinium esters. Separation and nonseparation assays have been devised, based on isoluminol and acridinium ester labels. The combination of the amplification properties of an enzyme and a CL or BL detection reaction provides a highly sensitive analytical system. Since 1983, CL and BL methods have been developed for many enzyme labels, e.g., alkaline phosphatase, glucose-6-phosphate dehydrogenase, horseradish peroxidase, Renilla luciferase, and xanthine oxidase. Currently, the most successful enzyme assays are the enhanced CL method for a peroxidase label involving a mixture of luminol, hydrogen peroxide, and an enhancer (e.g., p-iodophenol) and the direct CL method for alkaline phosphatase, with an adamantyl 1,2-dioxetane phenyl phosphate as substrate. Both systems are very sensitive (the detection limit for alkaline phosphatase when using the dioxetane reagent is 0.001 amol) and produce long-lived light emission (greater than 30 min), which is ideal for membrane applications in which light emission is detected with photographic film or a charge-coupled device camera.

Blotting, Western↗

A rapid electrochemical assay of lecithin in amniotic fluid using a fluoride ion-sensitive electrode.

An electrochemical method is described for the determination of lecithin in rat and human amniotic fluid. Choline is released from lecithin enzymatically by phospholipase D and the hydrogen peroxide released by the action of choline oxidase is quantitatively determined by peroxidase-catalyzed rupture of the covalent C-F bond of 4-fluorophenol. The concentration of F- ions in solutions is determined by a fluoride sensitive electrode from the resulting cell potential difference recorded before and 10 min after addition of a solution containing phospholipase D, choline oxidase and horseradish peroxidase. Lecithin levels in rat amniotic fluid increased from about 10 mumol/l on the 20th day of gestation to 80 mumol/l on day 21, which corresponds to the time of spontaneous delivery. In human amniotic fluid the lecithin concentrations determined with this new method parallel those already reported. They were approximately 10 to 50 mumol/l between the 15th and 18th weeks of gestation and increased from 5- to 7-fold between the 37th and 41st weeks of pregnancy. This method was only slightly influenced by the presence of blood or meconium contamination in the amniotic fluid.

Amniotic Fluid↗

A method for measuring H2O2 based on the potentiation of peroxidative NADPH oxidation by superoxide dismutase and scopoletin.

NADPH oxidation catalyzed by horseradish peroxidase is considerably increased by scopoletin and superoxide dismutase. These effects were used to develop a method for measuring H2O2 in a horseradish peroxidase, superoxide dismutase, and scopoletin system by measuring the NADPH oxidation rate. The optimal concentration of each reactant was determined. H2O2 could be detected and measured when it was present free in the medium or when it was produced by an H2O2-generating system, such as glucose-glucose oxidase or NADPH oxidase from thyroid plasma membranes. H2O2 was measured either by taking aliquots of the incubation medium or by placing NADPH directly in the medium and following the kinetics of NADPH oxidation. This latter approach required smaller amounts of biological material. In contrast to other methods, the H2O2 which is measured is regenerated. This method is 10 times more sensitive than the standard scopoletin method for H2O2 measurement and will detect a H2O2 production rate as low as 0.2 nmol per hour. The method is particularly suitable for biological systems in which small quantities of biological material are available.

Animals↗

Effects of cadmium and copper on antioxidant capacities, lignification and auxin degradation in leaves of pea (Pisum sativum L.) seedlings.

Twelve-day-old seedlings of pea were treated for four days by 20 and 100 microM of Cd(NO3)2 or CuSO4. In leaves, all treatments caused an increase in the lipoperoxidation product rate. However, 20 microM of Cu did not affect the growth. Moreover, except for 20 microM of Cu, the activity of unspecific peroxidases, used as stress marker, was enhanced in cell walls of metal-stressed plants. No change in the antioxidant capacities was observed in plants treated with 20 microM of metal. At this dose, the Cd-reduced growth could be associated to an elevation in the activities of IAA oxidase and of lignifying peroxidases. Increase of these latter, in concert with loss in antioxidant capacities, would be responsible for the growth diminution after exposure to 100 microM of metal. However, the activity of lignifying enzymes was not affected by 100 microM of Cu. The contribution of cell fractions to enzymatic responses to stress is emphasized.

Antioxidants↗

Flavin-containing polyamine oxidase is a hydrogen peroxide source in the oxidative response to the protein phosphatase inhibitor cantharidin in Zea mays L.

In this study, the specific contribution of polyamine oxidase (PAO), a hydrogen peroxide (H2O2)-producing enzyme, to the oxidative burst induced in maize mesocotyl by the phosphatase inhibitor cantharidin was examined. For this purpose, a pharmacological approach was applied using, either in vitro or in vivo, two strong inhibitors of maize PAO (MPAO), N-prenylagmatine (G3) and its structural analogue Ro5, as well as diphenyleneiodonium (DPI), an inhibitor of the phagocyte NAD(P)H oxidase. DPI was shown to be a good MPAO inhibitor in vitro. G3, Ro5, and DPI were very effective in inhibiting in vivo the extracellular accumulation of H2O2 that is released by mesocotyl segments upon spermidine supply. G3 and Ro5 did not show any inhibition in vitro of either horseradish peroxidase or barley oxalate oxidase. Moreover, G3 and Ro5 did not inhibit the extracellular accumulation of superoxide radical that is released in vivo upon NADH supply. G3, Ro5, and DPI strongly affected H2O2 production induced in maize mesocotyl by cantharidin. Histochemical localization of H2O2 in cantharidin-treated mesocotyl cross-sections revealed an increase of H2O2-specific staining in the epidermal and subepidermal tissues. The effect was also inhibited by G3 and DPI. Moreover, an increase in MPAO activity was observed in the same tissues upon cantharidin treatment. All these data suggest that G3 and Ro5 behave as powerful and selective inhibitors of MPAO activity either in vitro or in vivo and that MPAO activity contributes to a major part of the cantharidin-induced H2O2 synthesis in the apoplastic milieu of maize mesocotyl.

Agmatine↗

Fiberoptic biosensors based on chemiluminescent reactions.

The chemiluminescence of luminol in the presence of H2O2 has been exploited to develop fiberoptic biosensors associated with flow injection analysis systems. A chlorophenol sensor was developed based on the ability of certain halophenols to enhance the peroxidase-catalyzed luminol chemiluminescence. Horseradish peroxidase immobilized on a collagen membrane was used. Ten chlorophenols have been tested with this chemiluminescent-based sensor. The lower detection limit was obtained with 4-chloro-3-methylphenol and was equal to 0.01 microM. Electrochemiluminescent-based fiberoptic biosensors for glucose and lactate were also developed using glucose oxidase or lactate oxidase immobilized on polyamide membranes. In the presence of oxidase-generated H2O2, the light emission was triggered electrochemically by means of a glassy carbon electrode polarized at +425 mV vs a platinum pseudo-reference electrode. The detection limits for glucose and lactate were 150 and 60 pmol, respectively, and the dynamic ranges were linear from 150 pmol to 600 nmol and from 60 pmol to 60 nmol, respectively.

Biosensing Techniques↗

Recent advances on the molecular genetics of ligninolytic fungi.

This review highlights significant recent advances in the molecular genetics of white-rot fungi and identifies areas where information remains sketchy. The development of critical experimental tools such as genetic mapping techniques is described. A major portion of the text focuses on the structure, genomic organization and transcriptional regulation of the genes encoding peroxidases, laccases and glyoxal oxidase. Finally, recent efforts on heterologous expression of lignin-degrading enzymes are discussed.

Amino Acid Sequence↗

Effects of insulin on N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated production of reactive oxygen metabolites from normal human neutrophils.

OBJECTIVE AND DESIGN: To further understand the mechanisms behind defective neutrophil function in diabetes mellitus, the ability of insulin to affect the production and metabolism of reactive oxygen metabolites in normal human neutrophils was studied. MATERIALS AND METHODS: Neutrophil granulocytes from healthy adults were studied for their N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated production of reactive oxygen metabolites and release of myeloperoxidase or elastase following treatment with insulin. RESULTS: Preincubation of the neutrophils for 30 min with insulin (40-320 microU/ml), before activation with 1 microM fMet-Leu-Phe, revealed that the hormone at 80 and 160 microU/ml reduced the luminol-enhanced chemiluminescence without affecting the superoxide secretion. The insulin-induced reduction of the chemiluminescence response was reversed by the addition of exogenous peroxidase and was also paralleled by a reduced myeloperoxidase activity, with no effect on the elastase activity, in cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils. Superoxide dismutase removed the inhibitory effect of insulin on myeloperoxidase activity. CONCLUSIONS: These results suggest that elevated levels of insulin do not affect the NADPH-oxidase activity but, together with superoxide anions, interfere with myeloperoxidase availability and a subsequent myeloperoxidase-dependent generation of reactive oxygen metabolites in fMet-Leu-Phe-stimulated normal human neutrophils.

Adult↗

NADH oxidase of plasma membranes.

NADH oxidase is a cyanide-resistant and hormone-responsive oxidase intrinsic to the plasma membrane of both plant and animal cells. The activity has many unique characteristics that distinguish it from other oxidases and oxidoreductases of both organelles and internal membranes and from other oxidoreductases of the plasma membrane. Among these are resistance to inhibition by cyanide, catalase, superoxide dismutase, and phenylchloromercuribenzoate. Activity is stimulated by hormones and growth factors and inhibited by quinone analogs such as piercidin, the flavin antagonist atebrin, and growth inhibiting gangliosides such as GM3. In marked contact to the NADH-ferricyanide oxidoreductase of the plasma membrane, the NADH oxidase is activated by lysophospholipids and fatty acids, products of phospholipase A2 action, in a time-dependent manner suggestive of stabilization of an activated form of the enzyme. The hormone-responsive NADH oxidase of the plasma membrane is not a peroxidase and may function as a terminal oxidase to link transfer of electrons from NADH to oxygen at the plasma membrane. The functional significance of the NADH oxidase of the plasma membrane is unknown but some relationship to growth or growth control is indicated. In both animal and plant plasma membranes, the oxidase is activated by growth factors and hormones to which the cells or tissues of origin have functional hormone or growth factor receptors. In addition, substances that inhibit the oxidase, the associated transmembrane reductase or both, inhibit growth. In transformed cells and tissues, the hormone and growth factor responsiveness of the NADH oxidase is reduced or absent. With human keratinocytes which exhibit an increased sensitivity to the antiproliferative action of both retinoic acid and calcitriol, the NADH oxidase of the plasma membrane is strongly inhibited by these agents and shows the same increased sensitivity. If transfer of electrons from NADH to oxygen across or within the eukaryotic plasma membrane is an important aspect of growth or growth control, then the hormone- and growth factor-responsive NADH oxidase associated with the plasma membrane could be of fundamental importance. Because of its low basal activity, stimulation by growth factors and hormones, and the inhibition of growth in direct proportion to inhibition of the oxidase, the activity is a candidate as a rate-limiting step in the growth process. Completely unknown is the mechanism whereby NADH oxidation and growth or growth control may be coupled. This, together with further characterization of the activity and the mechanism of loss of control with neoplastic transformation, represent important challenges for future investigations.

Animals↗

[Enzymatic determination of total cholesterol with the Greiner Selective Analyzer (GSA-II) (author's transl)].

A fully enzymatic method to determine total cholesterol in serum is described. The method appears especially suitable for adaptation to discrete mechanical analyzers either of the single channel or the multi-channel type. The method uses the enzymes cholesterol esterase (EC 3.1.1.13), cholesterol oxidase (EC 1.1.3.6) and peroxidase (EC 1.11.1.7) with 4-aminophenazone and phenol as substrates in the indicator reaction. The method was adapted to the Greiner Selective Analyzer GSA-II. For this purpose the critical parameters of the reaction were intensively examined. The complete reagent is stable within the GSA II dispenser at 4 degrees C for at least 1 week. By omitting cholesterol oxidase in the blank reagent a sample bland and a partial reagent blank are obtained. Within a range up to 10.4 mmol/1 (4.0 g/l) the maximum colour is developed within 6 minutes. The calibration factor was stable for 4 months. The method allows absolute measurements. At concentrations between 2 and 4 mmol/1 within-batch precision ranged from 0.5 to 1.4%. Precision from day to day for the same control sera amounted to 2.8; 2.0; 2.7 and 2.0% for a period of 3 months. Examination of accuracy yielded satisfying results. Ascorbic acid in the physiological range did not alter results to a significant extent. Catalase or novaminesulfone added in vitro did not interfere. Optical interferences by bilirubin, hemoglobin or turbidity are compensated by a sample blank. A comparison of results with the enzymatic method of Roeschlau et al. (Z. Klin. Chem. Klin. Biochem. 12, 226 (1974)) yielded satisfactory agreement. The limits of detection of the present method can be lowered by a factor of 2.2 by replacing phenol by dihalogen phenols.

Autoanalysis↗

The immobilization of glucose oxidase onto radio-frequency plasma-modified poly(etherurethaneurea).

Glucose oxidase was covalently immobilized onto a radio-frequency plasma-modified poly(etherurethaneurea). Thin (90-100 nm) plasma-polymerized N-vinyl-2-pyrrolidone films were deposited onto poly(etherurethaneurea) films. Active sites for the immobilization were obtained via reduction with aqueous sodium borohydride and activation with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. Modified poly(etherurethaneurea) films were assayed for binding and activity of the immobilized glucose oxidase layer. The results of a modified radioimmunoassay and an 'immunochemical stain' indicated that washing in 900 ml of continuously stirred 2% sodium dodecyl sulfate, 2% Triton X-100, and 20 mM sodium phosphate, pH 7.0, for 24 h each at 4 degrees C was necessary to remove physically adsorbed glucose oxidase from the solid supports. An amperometric activity determination in 9 ml of well-stirred 20 mM sodium phosphate-0.1 M sodium chloride, pH 7.4, gave a qualitative demonstration of the activity of the immobilized enzyme on 18.75 cm2 of modified poly(etherurethaneurea) film. A colorometric activity determination using the coupled reaction with o-dianisidine and peroxidase indicated that glucose oxidase covalently immobilized on approximately 2.4 cm2 of modified poly(etherurethaneurea) film had an activity approximately equal to that of 13.4 nM glucose oxidase in 50 mM sodium acetate, pH 5.1, with a specific activity of approximately 32.0 U/mg at pH 5.1 and room temperature.

Biocompatible Materials↗

Conjugation of enzymes to immunoglobulins using dimaleimides.

A method is described for coupling enzymes to immunoglobulins using sulphydryl derivatives of the proteins and a dimaleimide which is relatively water-soluble. Parameters affecting the performance of the conjugates have been examined including level of sulphydryl incorporation, ratio of enzyme/immunoglobulin and nature of dimaleimide used. Peroxidase-immunoglobulin conjugates made by the dimaleimide method have been compared with those made by the periodate oxidation method and found to have a superior performance. Immunoglobulin has been linked to peroxidase (horseradish peroxidase, EC 1.11.1.7), glucose oxidase from Aspergillus niger, (EC 1.1.3.4), penicillinase from Bacillus cereus beta-lactamase I (EC 3.5.2.6), and beta-galactosidase from Escherichia coli (EC 3.2.1.23).

Immunoenzyme Techniques↗