Diabrotica speciosa (Germar) (Coleoptera: Chrysomelidae) is a major pest of common bean (Phaseolus vulgaris L.; Fabales: Fabaceae), and adults can defoliate plants during the whole crop cycle. This study was conducted to evaluate the resistance to D. speciosa in 16 common bean genotypes (14 landraces and 2 cultivars), through three different experiments. In the laboratory, choice and no-choice feeding tests were performed to evaluate the percentage of leaf consumption. In the greenhouse, plant height, numbers of leaves, percentage of injured leaves, percentage of injury per leaf, weight of seeds, and D. speciosa survival were evaluated. Furthermore, trichome density, levels of peroxidase (POD), superoxide dismutase (SOD), and protein content in common bean leaves were assessed. In the laboratory, the genotypes Chumbinho Branco, Dobalde, Manteigado, IPR Tuiuiú, and 90D Mouro were the least consumed by D. speciosa. In the greenhouse, the genotypes Dobalde, Manteigado, and IPR Tuiuiú expressed tolerance to the pest, which was associated with a higher plant height and/or unchanged POD and SOD levels and protein content following insect feeding, and no reduction in seed production. The landrace 90D Mouro exhibited antixenosis and tolerance to D. speciosa, observed as a lower leaf injury, higher trichome density, lower protein contents, higher SOD level and no reduction in seed weight. Overall, we have shown that antixenosis and tolerance can help overcome damages resulting from D. speciosa feeding, with emphasis on four common bean genotypes that may be useful in plant breeding programs aimed at controlling D. speciosa in common bean crops.

Ferric heme b (= ferric protoporphyrin IX = hemin) is an important prosthetic group of different types of enzymes, including the intensively investigated and widely applied horseradish peroxidase (HRP). In HRP, hemin is present in monomeric form in a hydrophobic pocket containing among other amino acid side chains the two imidazoyl groups of His170 and His42. Both amino acids are important for the peroxidase activity of HRP as an axial ligand of hemin (proximal His170) and as an acid/base catalyst (distal His42). A key feature of the peroxidase mechanism of HRP is the initial formation of compound I under heterolytic cleavage of added hydrogen peroxide as a terminal oxidant. Investigations of free hemin dispersed in aqueous solution showed that different types of hemin dimers can form, depending on the experimental conditions, possibly resulting in hemin crystallization. Although it has been recognized already in the 1970s that hemin aggregation can be prevented in aqueous solution by using micelle-forming amphiphiles, it remains a challenge to prepare hemin-containing micellar and vesicular systems with peroxidase-like activities. Such systems are of interest as cheap HRP-mimicking catalysts for analytical and synthetic applications. Some of the key concepts on which research in this fascinating and interdisciplinary field is based are summarized, along with major accomplishments and possible directions for further improvement. A systematic analysis of the physico-chemical properties of hemin in aqueous micellar solutions and vesicular dispersions must be combined with a reliable evaluation of its catalytic activity. Future studies should show how well the molecular complexity around hemin in HRP can be mimicked by using micelles or vesicles. Because of the importance of heme b in virtually all biological systems and the fact that porphyrins and hemes can be obtained under potentially prebiotic conditions, ideas exist about the possible role of heme-containing micellar and vesicular systems in prebiotic times.

Tyrosinases are a widespread family of multicopper oxidase enzymes. In our earlier work, we identified the presence of tyrosinases in lichenized Ascomycetes based on their substratum specificity, sensitivity to inhibitors and molecular mass. Here, we present a more detailed characterization of a tyrosinase from the lichen Lobaria pulmonaria. We also compare tyrosinase activity with the activities of laccases and peroxidases, the other redox enzymes present in this species. The importance of tyrosinases in lichen biology was studied by testing their role in melanin synthesis. Laboratory experiments clearly showed that tyrosinases from L. pulmonaria resemble those from other lichens and in free-living fungi. While the tyrosinases can metabolize the melanin precursor L-DOPA, L-DOPA can also be metabolized by peroxidases and laccases. A field experiment showed that exposing shade-adapted L. pulmonaria to normal solar radiation induces L-DOPA melanin synthesis. Synthesis occurred when lichens were exposed to either direct sunlight, or placed under a wavelength-neutral filter that slightly reduced overall light. In lichens receiving unfiltered sunlight, melanin synthesis was accompanied by increased laccase activity; by contrast, no changes in enzyme activity occurred in lichens placed under the wavelength-neutral filter. Melanization was reduced by placing lichens under filters that removed UV-B, and prevented by filters that removed both UV-A and UV-B. Removing UV-B had no effect on enzyme activity, whereas removing both UV-A and UV-B increased tyrosinase activity. Results from this study indicate that under some conditions laccases may be involved in melanin synthesis, but they provide no evidence for a role for tyrosinases in melanization. Although high tyrosinase activities are widespread in lichens, many questions on the role of this enzyme in lichen biology remain to be answered.

Introduction. Mature green vanilla pods accumulate 4-O-(3-methoxy-benzaldehyde)-β-D-glucoside (glucovanillin), which, upon hydrolysis by an endogenous b-glucosidase, liberates vanillin, the major aroma component of vanilla. Little is known on the spatial distribution of aroma-generating phenolics, and the enzymes responsible for their liberation (β-glucosidase) and oxidation (peroxidase). We report here quantitative data with respect to these three components in relation to the anatomy of the pod. Furthermore, the spatial progression of oxidation is shown. Materials and methods. Mature green vanilla pods were analyzed for their contents of phenolics (HPLC), and β-glucosidase and peroxidase activities by spectrophotometric techniques using p-nitrophenyl glucoside and vanillin as substrates, respectively. Lipids were examined under fluorescence microscopy after Nile red staining. Oxidation development was observed on transverse slices of pods. Results and discussion. Phenolics, and β-glucosidase and peroxidase activities showed gradients of increasing-decreasing concentrations from the stem to the blossom end of pods. The β-glucosidase activity is distributed in between the placentae, mesocarp, and trichomes in a [7 / 2 / 1] proportion while that of peroxidase shows a [38 / 1] ratio in the mesocarp and placentae, and was absent from trichomes. Oxidation begins from the blossom end in the placentae, progressively invading the mesocarp and moving towards the stem end. Conclusion. The green mature vanilla pod is spatially heterogeneous for its phenolics, and β-glucosidase and peroxidase activities, its placentae playing an important role in the liberation of vanillin and its subsequent oxidation.

Sepia officinalis produces a dark ink constituted of a suspension of melanin granules in a viscous colourless medium showing a large variability in composition. The examination of the spectra obtained by scanning electron microscopy of crude and the melanin-free ink showed slight variation in elemental composition related to the elimination of melanin substance after centrifugation. Ink elemental content varied also depending on the period of sampling. Temperature, light and oxygen can be considered as coagulation factors. Temperature around ambient temperature (e.g. 30°C) gave strong coagulation, while lower temperature (2–4°C), lack of oxygen and darkness greatly inhibited the ink coagulation process. Moreover, we showed that hydrogen peroxide activated the ink coagulation process and the coagulation rate depends on the amount of H2O2 added. Heat treatment (100°C for 5 minutes) of ink inhibited the coagulation. Interestingly, the addition of an adequate volume of fresh melanin-free ink to the heated sample activated significantly the coagulation process.

Electron paramagnetic resonance studies of high vigour (99% viable) and low vigour (38% viable) dry embryos of rice seeds (Oryza sativa L.) stored in a natural (warm and humid) environment were carried out. Loss in viability due to hot and humid conditions was found to be correlated with a decrease in free radical levels. The free radicals could be carbon-based and derived from quinones. Presence of the active oxygen scavenging enzyme, superoxide dismutase (EC 1.15.1.1) in embryos isolated from dry seeds showed a positive corrrelation with the state of vigour or viability. Anodic peroxidase (EC 1.11.1.7) activity in imbibed seeds also declined with the decline in vigour and viability. It is concluded that the deterioration of cells in the embryonic axis depends on the balance between free radical accumulation and the activity of active oxygen-scavenging enzymes which constitutes the active oxygen scavenging system (AOSS) during early imbibition. During prolonged storage under hot and humid conditions, cumulative effects of macromolecular damage due to oxidative chain products, compounded with the loss of enzyme activity, result in the final catastrophe, the death of the embryo.

At shedding, the moisture content (MC) of Avicennia marina (Forssk.) Vierh. propagules was 65% (fresh mass basis), and there was no significant difference in the MC of four tissues (hypocotyl, cotyledons, plumule and root primordia). Viability declined as the propagules were dried below 60% MC, so that only 40%of seeds were capable of germination at 54% MC. At 47% MC all the seeds had died. The four tissues dried at the same constant rate of 0.02 g water g dwt-1 h–1 throughout this range of MCs. There was no significant depletion of the free-radical-quenching mechanisms measured. In each tissue an organic free-radical was detected by electron paramagnetic resonance (EPR). In the plumule the amplitude of the signal increased by a further 50% at MCs where viability was lost, but there was no increase in the other tissues. There was a concurrent increase in the amount of tocopherol and the activity of superoxide dismutase in the plumule. Lipid peroxidation, assessed by the amount of thiobarbituric acid-reactive substances, also increased in advance of viability loss, suggesting that propagules were experiencing oxidative stress. However, lipid peroxidation decreased at 54–57% MC, where most seeds lost viability. The results presented are consistent with a propagule reacting to oxidative stress, but overtaken by more catastrophic physical damage.

This review discusses the production of reactive oxygen species (ROS) by lichens and their possible roles. All organisms produce ROS, and production is increased by many abiotic and biotic stresses. Intracellular ROS production is generally considered to be harmful, and a variety of enzymic and non-enzymic scavenging systems exist to detoxify them. However, extracellular ROS formation has been suggested to play ‘positive roles’, particularly in the response of organisms to stress. Given their high stress tolerance, it is rather surprising that studies on extracellular ROS production by lichens have just started. Surveys of a wide range of lichens have shown that constitutively high rates of extracellular superoxide production occur in the Suborder Peltigerineae, but production appears to be absent in other groups. In some members of the Peltigerineae ROS production is stimulated by desiccation and wounding. It seems probable that the enzymes that produce the superoxide are laccases, based on first the types of substrates that lichens can break down, and second the dependence of the breakdown of these substrates on pH, temperature and the presence of inhibitors. While much more work is needed, we suggest that physiological roles of extracellular ROS production will be found to include defence against pathogens, melanization, and lignin breakdown.

Antibodies to detect pectin in present investigations attached to distinct fibrils in vessel lumina. In carnation infected with an isolate of Fusarium oxysporum f.sp., labeling of pathogen cells also occurred; in a resistant cultivar (cv.), it was coincident with proximate pectin fibrils and linked to altered fungal walls, which was the opposite in the susceptible cv., indicating that hindrance of pathogen ability to degrade pectin may be related to resistance. Labeling of the fungus in culture was nil, except in media containing pectin, showing that pectin is not native to the pathogen. Labeling of fungal walls for cellulose in elm (inoculated with Ophiostoma novo-ulmi) and carnation also occurred, linked to adsorbed host wall components. The chitin probe often attached to dispersed matter, in vessel lumina, traceable to irregularly labeled fungal cells and host wall degradation products. With an anti-horseradish peroxidase probe, host and fungal walls were equally labeled, and with a glucosidase, differences of labeling between these walls were observed, depending on pH of the test solution. Fungal extracellular matter and filamentous structures, present in fungal walls, predominantly in another elm isolate (Phaeotheca dimorphospora), did not label with any of the probes used. However, in cultures of this fungus, extracellular material labeled, even at a distance from the colony margin, with an anti-fimbriae probe.

Background and objective The objective of this study was to determine the dose as well as the duration of exposure–dependent effects of methohexital on neutrophil [polymorphonuclear leucocyte (PMN)] free amino acid profiles and, in a parallel study, on PMN immune functions.

Methods Whole blood samples were taken from 20 volunteers and incubated with methohexital [0 (control), 3.6, 26, 130 and 260 μgmL−1] for 10, 30, 60 or 120 min. PMN amino acid profiles were documented using advanced PMN separation and highperformance liquid chromatography procedures. Superoxide anion (O2−) and hydrogen peroxide production (H2O2), and activity of released myeloperoxidase (MPO), were determined photometrically.

Results After methohexital, significant dose (≥26 μg mL−1) as well as duration of exposure–dependent (≥30 min) increases in histidine, isoleucine, leucine, valine, methionine, serine, glycine, threonine, and decreases in glutamine, glutamate, aspartate, asparagine, arginine, ornithine, citrulline, alanine and taurine were observed (P ≤0.05). Concerning PMN immune functions, methohexital significantly decreased O2−, H2O2 formation and MPO (≥26 μgmL−1, ≥30 min, P≤0.05).

Conclusions Altogether, there is significant relevance to the pharmacological regimens which enhance the supply of methohexital in whole blood. In regards to our results, we suggest that considerable changes in PMN ‘dynamic free amino acid pool’, for example induced by methohexital, may be one of the determinants in cell nutrition adversely affecting PMN metabolism. It is partially through its effect on the PMN free amino acid pool that maleficent pharmacological stress may have an unintentional influence on PMN immune functions.

Isozyme expression of nutrient-mobilizing and defence-related enzymes were examined in different functional components of intact Scots pine (Pinus sylvestris L.) ectomycorrhizal root systems developed in natural unsterilized forest humus. Scots pine seedlings colonized by Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) strains were grown on humus in two-dimensional Perspex[target] microcosms to allow development of complete ectomycorrhizal root systems. Soluble proteins from uncolonized short roots, whole mycorrhizal root tips or dissected mantle and core fractions, fungal strands and the outermost soil colonizing fine hyphae were subjected to native polyacrylamide gel electrophoresis and stained to detect esterase, acid phosphatase, peroxidase and different polyphenoloxidase isozyme activities. Tissue-specific esterase isozymes were detected in all components and most, including unique Hartig net isozymes, were of fungal origin. High fungal acid phosphatase activities were detected in mycorrhiza and fine hyphae of S. bovinus, supporting earlier findings of active phosphatase activity at the fungal interface in the Hartig net region and in the fine hyphal margins of extramatrical mycelium actively colonizing the humus. All compartments in the P. involutus mycorrhizosphere had weaker acid-phosphatase activities. Peroxidases formed a large part of the soluble-protein content in non-mycorrhizal short roots. The amount of peroxidases, on a tissue f. wt basis, was similar in mycorrhizal and non-mycorrhizal roots, but differential isozyme expression was detected, indicating a change in root peroxidase activities following mycorrhiza formation. The expression of DL-3,4-dihydroxyphenylalanine metabolizing polyphenol oxidase enzymes was reduced in mycorrhizas compared with non-mycorrhizal short roots. In the extramatrical mycelial components, fine hyphae expressed the highest polyphenoloxidase activity. P. involutus displayed some polyphenol-oxidizing isoenzymes not detected in S. bovinus systems. Laccase activity was not detected in the plant and fungal components examined. It is concluded that enzyme activities and isozyme expression are differentially regulated in the different functional components of Scots pine mycorrhizospheres.

The effect of methionine on longevity, antioxygenic enzymes and lipid peroxides in Callosobruchus maculatus was investigated using C. maculatus maintained on Vigna radiata seeds soaked in seven levels of methionine, 0, 1, 3, 5, 7, 9 and 12 mg/ml water. The life span and activities of catalase and peroxidase were higher in Callosobruchus maculatus infesting Vigna radiata seeds soaked in methionine as compared to their control cohorts. However, the lipid peroxide (malondialdehyde; MDA) levels generally remained unaltered. The present observation on the increased longevity in C. maculatus with methionine level is an extension of earlier findings on its anti-ageing effect in higher animals.