Mechanistic investigations of the novel non-heme vanadium bromoperoxidases. Evidence for singlet oxygen production

Three newly discovered non-heme bromoperoxidases isolated from marine algae were found to catalyze the production of singlet oxygen in reactions composed of the bromoperoxidase, hydrogen peroxide, and bromide. The bromoperoxidases studied were vanadium bromoperoxidase (V-BrPO) from Ascophyllum nodosum, native non-heme bromoperoxidase from Corallina vancouveriensis (which contains vanadium and iron), and the vanadium-reconstituted bromoperoxidase derivative from C. vancouveriensis. These enzyme systems generated near infrared emission, characteristic of singlet oxygen. The emission had a peak intensity near 1268 nm, was greatly increased in 2H2O-containing buffers, and was greatly decreased by the singlet oxygen quenchers, histidine and azide. The yield of singlet oxygen was approximately 80% of the theoretical yield. A unique feature of the non-heme bromoperoxidases distinct from the iron heme haloperoxidases, was the remarkable stability of the non-heme enzymes in the presence of singlet oxygen and oxidized bromine species. V-BrPO turned over multiple aliquots of 2 mM hydrogen peroxide without losing efficiency. In contrast, iron heme lactoperoxidase was completely inactivated after turnover of the first aliquot of 2 mM hydrogen peroxide, and iron heme chloroperoxidase was 50% deactivated. The profile of singlet oxygen formation by V-BrPO and the near stoichiometric yield of singlet oxygen suggest that the mechanism of singlet oxygen formation is the same as the mechanism of dioxygen formation determined by oxygen probe measurements.     

Mechanism of dioxygen formation catalyzed by vanadium bromoperoxidase. Steady state kinetic analysis and comparison to the mechanism of bromination

The steady state kinetic mechanism of the bromide-assisted disproportionation of hydrogen peroxide, forming dioxygen, catalyzed by vanadium bromoperoxidase has been investigated and compared to the mechanism of monochlorodimedone (MCD) bromination under conditions of 0.0125-6 mM H2O2, 1-500 mM Br-, and pH 4.55-6.52. Under these conditions, 50 microM MCD was sufficient to inhibit at least 90% of the dioxygen formation during MCD bromination. The rate data is consistent with a substrate-inhibited Bi Bi Ping Pong mechanism, in which the substrate bromide, is also an inhibitor at pH 4.55 and 5.25, but not at pH 5.91 and 6.52. The kinetic parameter KmBr, KmH2O2, KisBr, and KiiBr determined for the reactions of bromide-assisted disproportionation fo hydrogen peroxide and MCD bromination are similar, indicating that the mechanisms of both reactions occur via the formation of a common intermediate, the formation of which is rate-limiting. Fluoride is a competitive inhibitor with respect to hydrogen peroxide in both reactions at pH 6.5. At high concentrations of hydrogen peroxide, the bromide-assisted disproportionation of hydrogen peroxide occurs during the bromination of MCD. The sum of the rates of MCD bromination and dioxygen formation during MCD bromination is equal to the rate of dioxygen formation in the absence of MCD. The apportionment of the reaction through the MCD bromination and dioxygen formation pathways depends on pH, with much lower hydrogen peroxide concentrations causing significant dioxygen formation at higher pH.     

The effect of nitrogen fertilization on the phenology of roots in a barrier island sand dune community

Little work has been done on the phenology of root growth and senescence largely due to methodological difficulties. The application of minirhizotron technology has enabled the tracking of individual roots through an entire growing season. As a result, direct measures of mortality, root growth, and an analysis of cohorts can be obtained. This study examined the belowground response of vegetation in a nutrient limited system to nitrogen addition. Small plots on a 36 year old dune on Hog Island, a barrier island in the Virginia Coast Reserve Long Term Ecological Research Site, were fertilized with nitrogen. Minirhizotron tubes were installed in each fertilized and control plot. Each tube was sampled monthly for nine months, March through October of 1992. Root length density increased throughout the growing season with the greatest root length density in the top 20 cm of the soil profile. The fertilized plots had greater root length densities (14.1 mm cm^-2) than the unfertilized plots (2.9 mm cm^-2). There was no significant depth × treatment interaction. Root mortality did not significantly change with fertilization. The largest loss of roots for a cohort occurred within the first month. The dune grassland community did not respond to fertilization with large changes in root distribution or increases in mortality in this study.     

Thermodynamics of strecker synthesis in hydrothermal systems

Submarine hydrothermal systems on the early Earth may have been the sites from which life emerged. The potential for Strecker synthesis to produce biomolecules (amino and hydroxy acids) from starting compounds (ketones, aldehydes, HCN and ammonia) in such environments is evaluated quantitatively using thermodynamic data and parameters for the revised Helgeson-Kirkham-Flowers (HKF) equation of state. Although there is an overwhelming thermodynamic drive to form biomolecules by the Strecker synthesis at hydrothermal conditions, the availability and concentration of starting compounds limit the efficiency and productivity of Strecker reactions. Mechanisms for concentrating reactant compounds could help overcome this problem, but other mechanisms for production of biomolecules may have been required to produce the required compounds on the early Earth. Geochemical constraints imposed by hydrothermal systems provide important clues for determining the potential of these and other systems as sites for the emergence of life.     

Real-time PCR for detection and quantification,and histological characterization of <Emphasis Type="Italic">Neonectria ditissima</Emphasis> in apple trees

Key message

We designed a pair of primers from a region of the β-tubulin gene to detect and quantify Neonectria ditissima in wood of some infected apple cultivars, and optimized light microscopy to study fungal-plant interactions.

Abstract

Neone ctria ditissima, the causal pathogen of fruit tree canker, is a sordariomycete fungus that affects apple orchards, especially in north-western Europe. To prevent serious disease epidemics, an accurate, rapid, and sensitive method for detection of N. ditissima is needed for pathogen identification. A quantitative real-time PCR (qPCR) assay was developed for both detection and quantification of this pathogen in infected apple cultivars. Several primer sets were designed from regions of the β-tubulin gene. One primer set passed several validation tests, and the melting curve confirmed species-specific amplification of the correct product. In addition, the N. ditissima biomass could be detected at variable amounts in samples from the infection sites of six different cultivars, with ‘Aroma’ having the lowest amount of N. ditissima biomass and ‘Elise’ the highest. To complement the qPCR results, tissue from detached shoots and 1-year-old trees of ‘Cox’s Orange Pippin’ (susceptible) and ‘Santana’ (partially resistant) was used in a histopathology study. In both detached shoots and trees, fungal hyphae were found in cells of all tissues. No qualitative differences in the anatomy of the infected samples were observed between the cultivars. In the detached shoot experiment, both cultivars were affected but differences in the rate of disease progression suggest that the partially resistant cultivar could resist the fungus longer. The qPCR assay developed in our study produced reproducible results and can be used for detection of N. ditissima in infected trees.

     

Effect of Sodium Fluorescein and Plating Medium on Recovery of Irradiated Escherichia coli and Serratia marcescens from Aerosols

Irradiation of aerosols of either Escherichia coli or Serratia marcescens with simulated solar (xenon) radiation caused a significant decrease in viability. When sodium fluorescein was employed to determine the physical loss of organisms from the aerosol, an additional adverse effect upon survival was noted. The decay curves indicated that at least two mechanisms of inactivation were operative, one due to aerosolization, the other to irradiation. After collection from aerosols, both species of microorganisms grew better on blood agar base than on Casitone agar, but this finding did not appear to be related to the effect of irradiation.     

A CYTOLOGICAL STUDY OF THE RELATION OF THE CORTICAL REACTION TO SUBSEQUENT EVENTS OF FERTILIZATION IN URETHANE-TREATED EGGS OF THE SEA URCHIN, ARBACIA PUNCTULATA

Eggs of the sea urchin, Arbacia punctulata, treated with 3% urethane for 30 sec followed by 0.3% urethane and inseminated are polyspermic and fail to undergo a typical cortical reaction. Upon insemination the vitelline layer of urethane-treated eggs either does not separate or is raised only a short distance from the oolemma. 1–6 min after insemination, almost all of the cortical granules remain intact and are dislodged from the plasmalemma. Later (6 min to the two-cell stage) some cortical granules are released randomly along the surface of the zygote. Not all zygotes show the same degree of cortical granule dehiscence; most of them experience little if any granule release whereas others demonstrate considerably more. The thickness of the hyaline layer appears to be directly related to the number of cortical granules released. Subsequent to pronuclear migration, several male pronuclei become associated with the female pronucleus. Later the male and female pronuclear envelopes contact and the outer and the inner laminae fuse, thereby forming the zygote nucleus. The male pronuclei remaining in the cytoplasm increase in size and progressively migrate to, and fuse with, the zygote nucleus. By 60 min some zygotes appear to contain only one large zygote nucleus which subsequently enters mitosis. Other zygotes possess a number of male pronuclei which remain unfused, and later these pronuclei along with the zygote nucleus undergo mitosis. There does not appear to be a direct relation between the number of cortical granules a zygote possesses and the above mentioned dichotomy.