2-hydroxy-3-phenylpropanoic acid suppressed the growth of Alternaria alternata through damaging cell membrane integrity and modulating reactive oxygen species metabolism

Black spot rot caused by Alternaria alternata is one of the major postharvest disease of apple fruit during logistic. This study evaluated in vitro inhibitory effect of 2-hydroxy-3-phenylpropanoic acid (PLA) at various concentrations on A. alternata and the possible mechanisms involved in its action. Results showed that different concentrations of PLA inhibited conidia germination and mycelial growth of A. alternata in vitro, and 1.0 g L⁻¹ was the lowest effective concentration to suppress A. alternata growth. Moreover, PLA significantly reduced relative conductivity and increased malondialdehyde and soluble protein contents. PLA also increased H₂O₂ and dehydroascorbic acid contents, but reduced ascorbic acid content. Additionally, PLA treatment inhibited catalase, ascorbate peroxidase, monodehydroascorbate acid reductase, dehydroascorbic acid reductase and glutathione reductase activities, whereas promoted superoxide dismutase activity. All these findings suggest that the possible mechanisms involved in the inhibitory effect of PLA on A. alternata included damaging the cell membrane integrity to cause electrolyte leakage and destroying reactive oxygen species balance.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis

The glutathione redox couple (GSH/GSSG) and hydrogen peroxide (H₂O₂) are central to redox homeostasis and redox signaling, yet their distribution within an organism is difficult to measure. Using genetically encoded redox probes in Drosophila, we establish quantitative in vivo mapping of the glutathione redox potential (E_GSH) and H₂O₂ in defined subcellular compartments (cytosol and mitochondria) across the whole animal during development and aging. A chemical strategy to trap the in vivo redox state of the transgenic biosensor during specimen dissection and fixation expands the scope of fluorescence redox imaging to include the deep tissues of the adult fly. We find that development and aging are associated with redox changes that are distinctly redox couple-, subcellular compartment-, and tissue-specific. Midgut enterocytes are identified as prominent sites of age-dependent cytosolic H₂O₂ accumulation. A longer life span correlated with increased formation of oxidants in the gut, rather than a decrease.     

Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension

The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O₂) or high (20% O₂) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoy's 5A medium for 6 days at 37 °C, 5% CO₂, and 5% or 20% O₂. On Days 4 and 6, the fragments were restained by adding NR to the McCoy's medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was observed (P < 0.001), resulting in an average growth of 11.82 ± 0.81 μm (5% O₂) and 11.78 ± 1.06 μm (20% O₂) on Day 4 and 20.94 ± 1.24 μm (5% O₂) and 19.12 ± 1.36 μm (20% O₂) on Day 6 compared with Day 0. No significant differences in follicle growth rate or stage transition could be observed between 5% and 20% O₂ (P > 0.05). In conclusion, after repeated NR staining, we could not find a difference between low and high oxygen tension in terms of follicle viability, stage transition, or growth. Therefore, under our culture conditions follicle dynamics are not determined by the oxygen tension in combination with quality assessment protocols using repeated NR staining.     

Ethyl gallate attenuates acute lung injury through Nrf2 signaling

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is the clinical syndrome of persistent lung inflammation caused by various direct and indirect stimuli. Despite advances in the understanding of disease pathogenesis, few therapeutic have emerged for ALI/ARDS. Thus, in the present study we evaluated the therapeutic potential of ethyl gallate (EG), a plant flavanoid in the context of ALI using in vivo (BALB/c) and in vitro models (human monocytes). Our in vivo data supports the view that EG alleviates inflammatory condition in ALI as significant reduction in BALF neutrophils, ROS, proinflammatory cytokines and albumin levels were observed with the single i.p of EG post LPS exposure. Also, histochemical analysis of mice lung tissue demonstrated that EG restored LPS stimulated cellular influx inside the lung airspaces. Unraveling the mechanism of action, our RT-PCR and western blot analysis suggest that enhanced expression of HO-1 underlies the protective effect of EG on ROS level in mice lung tissue. Induction of HO-1 in turn appears to be mediated by Nrf2 nuclear translocation and consequent activation and ablation of Nrf2 activity through siRNA notably abrogated the EG induced protective effect in LPS induced human monocytes. Furthermore, our results indicate that EG generated moderate amounts of H₂O₂ could induce Nrf2 translocation in the in vitro systems. However, given the insignificant amount of H₂O₂ recorded in the injected material in the in vivo system, additional mechanism for EG action could not be excluded. Nevertheless our results highlight the protective role of EG in ALI and provide the novel insight into its usefulness as a therapeutic tool for the treatment of ALI.     

Development of a benzene vapour sensor utilizing chemiluminescence on Y2O3

Chemiluminescence (CL) was observed when benzene vapour passed through the surface of Y₂O₃, TiO₂, Y₂O₃–V₂O₅, TiO₂–Y₂O₃, Y₂O₃–Cr₂O₃, Y₂O₃–Al₂O₃ and TiO₂–Al₂O₃, with air as the carrier gas. The strongest CL intensity was found with Y₂O₃ as the catalyst. A novel benzene sensor based on this kind of CL was developed. Quantitative analysis was performed at the wavelength of 425 nm. Under optimal conditions, CL intensity was directly proportional to the concentration of benzene vapour. The linear range was 4–7018 mg/m³ (r = 0.9981, n = 11), with a detection limit of 1 mg/m³ (the signal:noise ratio was 3). This gas sensor can work continuously for >80 h and has been successfully applied to the real‐time determination of benzene vapour. Copyright © 2008 John Wiley & Sons, Ltd.     

Quantitative profiling of prostaglandins as oxidative stress biomarkers in vitro and in vivo by negative ion online solid phase extraction – Liquid chromatography–tandem mass spectrometry

Free radical-mediated oxidation of arachidonic acid to prostanoids has been implicated in a variety of pathophysiological conditions such as oxidative stress. Here, we report on the development of a liquid chromatography–mass spectrometry method to measure several classes of prostaglandin derivatives based on regioisomer-specific mass transitions down to levels of 20 pg/ml applied to the measurement of prostaglandin biomarkers in primary hepatocytes. The quantitative profiling of prostaglandin derivatives in rat and human hepatocytes revealed the increase of several isomers on stress response. In addition to the well-established markers for oxidative stress such as 8-iso-prostaglandin F_2α and the prostaglandin isomers PE₂ and PD₂, this method revealed a significant increase of 15R-prostaglandin D₂ from 236.1 ± 138.0 pg/1E6 cells in untreated rat hepatocytes to 2001 ± 577.1 pg/1E6 cells on treatment with ferric NTA (an Fe³⁺ chelate with nitrilotriacetic acid causing oxidative stress in vitro as well as in vivo). Like 15R-prostaglandin D₂, an unassigned isomer that revealed a more significant increase than commonly analyzed prostaglandin derivatives was identified. Mass spectrometric detection on a high-resolution instrument enabled high-quality quantitative analysis of analytes in plasma levels from rat experiments, where increased concentrations up to 23-fold change treatment with Fe(III)NTA were observed.     

Effect of talc-formulated entomopathogenic fungus Beauveria against leaffolder (Cnaphalocrosis medinalis) in rice

Thirteen Beauveria strains were isolated from the soil and infected insects. Among the various isolates, B₂ isolate (Arachalore) showed a higher percentage of mortality against C. medinalis (73.3%) under in vitro conditions. Conidial concentration of 1 × 10⁸ of the B2 strain registered maximum mortality of 76.7%. The least LT₅₀ value of 4.4 days was registered in B2 isolate with the spore concentration of 1 × 10⁸ and the LC₅₀ value was 3.4 × 10⁴. Beauveria strains altered the feeding behavior of C. medinalis, reduced the pupal weight, prolonged the pupation period, malformed the pupa and adult under in vitro. The efficacy of the talc-based bioformulation of Beauveria (B2) strain was tested as seed treatment + seedling dip + soil application + foliar spray against rice leaffolder under in vitro and greenhouse conditions. The percentage damage was significantly less (5.5) in B2 as compared to untreated healthy control (25.8). In addition, the same treatment increased the activities of defense-related enzymes, namely peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, chitinase, and phenolics in rice.     

Molecular Characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica juncea</Emphasis> Cu/Zn-Superoxide Dismutases Reveals Their Regulation of Shoot Regeneration

Superoxide dismutases (SODs) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals (O₂^-) to molecular oxygen (O₂) and hydrogen peroxide (H₂O₂). In this study we characterized an Arabidopsis thaliana CuZnSOD (CSD1), a close ortholog of a previously identified Brassica juncea CuZnSOD (MSOD1). CSD1 and other two homologs CSD2 and CSD3 were spatially regulated in Arabidopsis, and CSD1 exhibited distinct expression patterns in response to different stress treatments. To investigate the in vivo function of SOD, transgenic Arabidopsis plants, expressing sense and antisense MSOD1 RNAs, were generated and those with altered SOD activity were selected for further characterization. Although SOD transgenic plants exhibited normal phenotypes, the shoot regeneration response in transgenic explants was significantly affected by the modulated SOD activity and the corresponding H₂O₂ levels. Transgenic explants with downregulated SOD activity were poorly regenerative, whereas those with upregulated SOD activity were highly regenerative. These results suggest that shoot regeneration in vitro is regulated by the SOD activity.     

Chemical quenching of singlet oxygen by plastoquinols and their oxidation products in Arabidopsis

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen (¹O₂) formed during high light stress in higher plants. Although quenching of ¹O₂ by prenylquinols has been previously studied, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol‐9 (PQH₂‐9) in chemical quenching of ¹O₂ was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH₂‐9 and plastochromanol‐8 biosynthesis. In this work, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of ¹O₂ was associated with consumption of PQH₂‐9 and formation of its various oxidized forms. Oxidation of PQH₂‐9 by ¹O₂ leads to plastoquinone‐9 (PQ‐9), which is subsequently oxidized to hydroxyplastoquinone‐9 [PQ(OH)‐9]. We provide here evidence that oxidation of PQ(OH)‐9 by ¹O₂ results in the formation of trihydroxyplastoquinone‐9 [PQ(OH)₃‐9]. It is concluded here that PQH₂‐9 serves as an efficient ¹O₂ chemical quencher in Arabidopsis, and PQ(OH)₃‐9 can be considered as a natural product of ¹O₂ reaction with PQ(OH)‐9. The understanding of the mechanisms underlying ¹O₂ chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.     

In vitro sucrose concentration affects growth and acclimatization of <Emphasis Type="Italic">Alocasia amazonica</Emphasis> plantlets

Plantlets of Alocasia amazonica were regenerated on the MS medium supplemented with different concentrations (0–9%) of sucrose. An absence of sucrose in the growth medium induced generation of leaves, however, it decreased multiplication. On contrary, sucrose supply of 6% or 9% enhanced multiplication but hampered photoautotrophic growth (generation of leaves). Increasing sucrose supply also increased sugars and starch content and number of stomata and decreased water potential and size of stomata during in vitro growth period. During ex vitro acclimatization, shoot length, root length, leaf number and root number of Alocasia plantlets grown with 3% sucrose, were found to be better among the other studied sucrose concentrations. Under ex vitro acclimatization, number of stomata, contents of various carbohydrates in the leaves were increased but size of stomata decreased with increasing sucrose supply during in vitro growth period. Moreover, water potential of leaves of plantlets, which have been grown with a sucrose concentration other than 3%, was decreased. During in vitro growth, net CO₂ assimilation rate (P_N), transpiration (E), stomatal conductance (g_s) and variable fluorescence to maximum fluorescence ratio (Fv/Fm) were unaffected, however, during acclimatization these were changed and maximum P_N, E, and g_s were observed in the plantlets micropropagated with 3% sucrose. Fv/Fm was decreased severely in the plantlets micropropagated with 6% sucrose during acclimatization. Thus a sucrose concentration of 3% in the medium is appeared to be better among studied concentrations for both in vitro growth and ex vitro acclimatization of A. amazonica plantlets.     

Peaks of in situ N2O emissions are influenced by N2O‐producing and reducing microbial communities across arable soils

Agriculture is the main source of terrestrial N₂O emissions, a potent greenhouse gas and the main cause of ozone depletion. The reduction of N₂O into N₂ by microorganisms carrying the nitrous oxide reductase gene (nosZ) is the only known biological process eliminating this greenhouse gas. Recent studies showed that a previously unknown clade of N₂O‐reducers (nosZII) was related to the potential capacity of the soil to act as a N₂O sink. However, little is known about how this group responds to different agricultural practices. Here, we investigated how N₂O‐producers and N₂O‐reducers were affected by agricultural practices across a range of cropping systems in order to evaluate the consequences for N₂O emissions. The abundance of both ammonia‐oxidizers and denitrifiers was quantified by real‐time qPCR, and the diversity of nosZ clades was determined by 454 pyrosequencing. Denitrification and nitrification potential activities as well as in situ N₂O emissions were also assessed. Overall, greatest differences in microbial activity, diversity, and abundance were observed between sites rather than between agricultural practices at each site. To better understand the contribution of abiotic and biotic factors to the in situ N₂O emissions, we subdivided more than 59,000 field measurements into fractions from low to high rates. We found that the low N₂O emission rates were mainly explained by variation in soil properties (up to 59%), while the high rates were explained by variation in abundance and diversity of microbial communities (up to 68%). Notably, the diversity of the nosZII clade but not of the nosZI clade was important to explain the variation of in situ N₂O emissions. Altogether, these results lay the foundation for a better understanding of the response of N₂O‐reducing bacteria to agricultural practices and how it may ultimately affect N₂O emissions.     

Kinetics of prolonged photoinhibition revisited: photoinhibited Photosystem II centres do not protect the active ones against loss of oxygen evolution

Photoinhibition of Photosystem II (PSII) in lincomycin-treated leaves begins as a first-order reaction, but fluorescence measurements have suggested that after prolonged illumination, the number of active PSII centres stabilizes to 15–20% of control. The stabilization has been interpreted to indicate that photoinhibited PSII centres protect the remaining active centres against photoinhibition (Lee, Hong and Chow, Planta 212:332–342, 2001). In an attempt to study the mechanism of this protection, we measured the reaction kinetics of photoinhibition in lincomycin-treated pumpkin (Cucurbita pepo L.) and pepper (Capsicum annuum L.) leaves in vivo. The light-saturated rate of PSII oxygen evolution, assayed from thylakoids and isolated from the treated leaves, was used as a direct measure of the number of remaining active PSII centres, and the fluorescence parameters F _V/F _M and (F _V/F _M)/F ₀ (=1/F ₀ − 1/F _M) were measured for comparison. To our surprise, no stabilization of PSII activity was observed and photoinhibition followed first-order kinetics until PSII activity had virtually declined to zero. A series of in vitro experiments was carried out to see whether stabilization of PSII activity occurs if a particular combination of light intensity and wavelength range is applied, or if a specific PSII preparation is used as experimental material. The results of the in vitro experiments confirmed the in vivo result about persistent first-order kinetics. We conclude that photoinhibited PSII centres offer no measurable protection against photoinhibition.     

Nobiletin protects human retinal pigment epithelial cells from hydrogen peroxide–induced oxidative damage

Nobiletin (3′,4′,5,6,7,8‐hexamethoxyflavone), a dietary polymethoxylated flavonoid found in Citrus fruits, has been reported to have antioxidant effect. However, the effect of nobiletin on human retinal pigment epithelium (RPE) cells induced by hydrogen peroxide (H₂O₂) is still unclear. Therefore, we investigated the protective effect of nobiletin against H₂O₂‐induced cell death in RPE cells. Our results demonstrated that nobiletin significantly increased cell viability from oxidative stress. Nobiletin inhibited H₂O₂‐induced ROS production and caspase‐3/7 activity in ARPE‐19 cells. Furthermore, nobiletin significantly increased Akt phosphorylation in ARPE‐19 cells exposed to H₂O₂. Meanwhile, LY294002, an inhibitor of PI3K/Akt, abolished the protective effect of nobiletin against H₂O₂‐induced decreased cell viability and increased caspase‐3/7 activity in ARPE‐19 cells. In summary, these data show that nobiletin protects RPE cells against oxidative stress through activation of the Akt‐signaling pathway. Thus, nobiletin should be an oxidant that attenuates the development of age‐related macular degeneration.     

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4)

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O₂-based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm² membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 μm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m⁻²·d⁻¹ maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.     

A Simple and Sensitive SPRS Method for Trace H2O2 Based on the TiO2+ Complex and Gold Nanoparticles Aggregation Reactions

In the medium of H₂SO₄ and in the presence of TiO²⁺, gold nanoparticles in size of 10 nm exhibited a weak surface plasmon resonance scattering (SPRS) peak at 775 nm. Upon addition of trace H₂O₂, the yellow complex [TiO(H₂O₂)]²⁺ formed that cause the gold nanoparticles aggregations to form bigger gold nanoparticle clusters in size of about 900 nm, and the SPRS intensity at 775 nm (I) enhanced greatly. The enhanced intensity ΔI was linear to the H₂O₂ concentration in the range of 0.025–48.7 μg/mL, with a detection limit of 0.014 μg/mL H₂O₂. This SPRS method was applied to determining H₂O₂ in water samples with satisfactory results.     

Steroid hormones promote bovine oocyte growth and connection with granulosa cells

Many approaches have been investigated for growing oocytes in vitro in mammals. To support oocyte growth in vitro, the culture systems must meet certain conditions for maintaining connections between oocytes and surrounding granulosa cells. The aims of this study were to determine the effects of combinations of 17β-estradiol (E₂) and androstenedione (A₄) on in vitro growth of bovine oocytes and to determine the number of connections between the oocyte and granulosa cells. Oocyte–granulosa cell complexes (OGCs) collected from early antral follicles (0.4−0.7 mm in diameter) were cultured for 14 days in a medium with different concentrations of E₂ and A₄, either alone or in combinations. We then assessed the number of transzonal projections (TZPs), which extend from granulosa cells through the zona pellucida to the oolemma. During in vitro growth culture, OGC structures were maintained in the medium with steroid hormones. The mean diameter of oocytes grown in the medium with both E₂ and A₄ was increased from 95.8 μm to around 120 μm, larger than oocytes grown without steroid hormones (109.9 μm) and similar in size to in vivo fully grown oocytes (119.4 μm) from 4- to 6-mm antral follicles. In subsequent in vitro maturation culture (22 hours), 30% (12 of 40) and 34% (14 of 41) of oocytes grown with E₂ or A₄ alone, respectively, matured to metaphase II; meanwhile, oocytes grown with a combination of E₂ and A₄ matured to metaphase II at a high rate (58%, 23 of 40). Growing oocytes isolated from early antral follicles had many uniformly distributed TZPs throughout the zona pellucida. After 14 days of culture, there was a significant decrease in the number of TZPs in oocytes grown without steroid hormones, whereas the number of TZPs was maintained in oocytes grown with steroid hormones. In particular, oocytes grown with E₂ alone or with a combination of E₂ and A₄ had numbers of TZPs similar to oocytes before growth culture. In conclusion, a combination of E₂ and A₄ maintained the connections between oocytes and granulosa cells during in vitro growth culture of bovine oocytes for 14 days, resulting in the complete oocyte growth and the acquisition of meiotic competence in more than half the oocytes.