Chemiluminescence analysis of oil oxidizing bacteria Actinetobacter calcoaceticus extracts: effects of the extracts on pSoxS-lux biosensor

A comparative H2O2-luminol- and Fe(II)-induced chemiluminescence analysis of extracts of two strains of marine oil oxidizing bacteria Actinetobacter calcoaceticus cultivated either in the presence or absence of oil was carried out. Effects of these extracts on E. coli MG1655 biosensor (pSoxS-lux) were studied. Activation of H2O2-induced chemiluminescence in the presence of oil was observed. This suggests activation of free radical lipid peroxidation. Aqueous extracts of microorganisms cultivated in the presence of oil were shown to activate reactive oxygen species production (ROS) in Fe(II)-induced chemiluminescence reaction mixture. Acetone-ethanol extracts induced antioxidative systems of both strains. Chemiluminescence analysis in a biological system carried utilizing E. coli MG1655 (pSoxS-lux) revealed that aqueous extracts of the strains cultivated in the absence of oil contained potential antioxidants.     

Motility influences biofilm architecture in Escherichia coli

Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms (∼43 μm thick, 21 to 34% surface coverage). JM109, DH5α, and MG1655 motA formed intermediate biofilms (∼13 μm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5α) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).     

Construction of the new <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 MG 1655 novel strain with improved growth characteristics for application in metabolic engineering

MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The “polar effect” of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val^S phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph ^wt from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying λattL-Cm^R-λattR and transduced with phage P1 into MG1655; later, the Cm^R marker was removed with the use of λXis/Int recombinase. Parallel to this procedure, a spontaneous Val^R mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of L-Val (50 μg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val^R strain had been generated in the region of the identified ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val^R phenotype in the obtained strain, was transferred to MG1655-rph ^wt using cotransduction, by analogy to the transfer of rph ^wt. Evaluation of rates of aerobically growing cells (μ, hour-1) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655,MG1655-rph ^wt, and MG1655-(rph ^wt, ilvG-15), respectively.     

Engineering of a robust Escherichia coli chassis and exploitation for large-scale production processes

In large-scale bioprocesses microbes are exposed to heterogeneous substrate availability reducing the overall process performance. A series of deletion strains was constructed from E. coli MG1655 aiming for a robust phenotype in heterogeneous fermentations with transient starvation. Deletion targets were hand-picked based on a list of genes derived from previous large-scale simulation runs. Each gene deletion was conducted on the premise of strict neutrality towards growth parameters in glucose minimal medium. The final strain of the series, named E. coli RM214, was cultivated continuously in an STR-PFR (stirred tank reactor – plug flow reactor) scale-down reactor. The scale-down reactor system simulated repeated passages through a glucose starvation zone. When exposed to nutrient gradients, E. coli RM214 had a significantly lower maintenance coefficient than E. coli MG1655 (Δm_s = 0.038 g_Glucose/g_CDW/h, p < 0.05). In an exemplary protein production scenario E. coli RM214 remained significantly more productive than E. coli MG1655 reaching 44% higher eGFP yield after 28 h of STR-PFR cultivation. This study developed E. coli RM214 as a robust chassis strain and demonstrated the feasibility of engineering microbial hosts for large-scale applications.     

Bacterial quorum sensing and cell surface electrokinetic properties

The hypothesis tested in this paper is that quorum sensing influences the microbial surface electrokinetic properties. Escherichia coli MG1655 and MG1655 LuxS- mutant (lacking quorum-sensing gene for Autoinducer synthase AI-2) were used for this study. AI-2 production (or lack of) in both strains was analyzed using the Vibrio harveyi bioassay. The levels of extracellular AI-2 with and without glucose in the growth medium were consistent with previously published work. The surface electrokinetic properties were determined for each strain of E. coli MG1655 by measuring the electrophoretic mobility using a phase amplitude light-scattering (PALS) Zeta potential analyser. The findings show that the surface charge of the cells is dependent upon the stage in the growth phase as well as the ability to participate in quorum sensing. In addition, significant differences in the electrophoretic mobility were observed between both strains of E. coli. These findings suggest that quorum sensing plays a significant role in the surface chemistry of bacteria during their growth.     

Anaerobic synthesis of succinic acid by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strains with activated NAD<Superscript>+</Superscript>-reducing pyruvate dehydrogenase complex

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD⁺-reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acid from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids through deletion of the genes ackA, pta, poxB, and ldhA (SGM0.1) in E. coli MG1655 strain and by additional introduction of the Bacillus subtilis pyruvate carboxylase (SGM0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 P_L-aceEF-lpdA and SGM0.1 P_L-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P_L-aceEF-lpdA and SGM0.1 P_L-aceEF-lpdA [pPYC] exceeded the corresponding yields of control strains by 2 and 33% in the absence and by 9 and 26% in the presence in media of HCO₃⁻ ion. It is concluded that an increase in the succinic acid production by strain SGM0.1 P_L-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive branch of the tricarboxylic acid cycle, is caused by activation of the glyoxylate shunt.     

2D [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C] NMR study of carbon fluxes during glucose utilization by <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells-glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655Δ(edd-eda), MG1655Δ(zwf, edd-eda), and MG1655Δ(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.     

Antigenotoxic activity of biologically active substances from <Emphasis Type="Italic">Inula britannica</Emphasis> and <Emphasis Type="Italic">Limonium gmelini</Emphasis>

The antigenotoxic and antioxidant activities of biologically active substances of extracts from Inula britannica L. and Limonium gmelinii (Willd.) Kuntze in E. coli strains MG1655 (pColD-lux), MG1655 (pSoxS-lux), and MG1655 (pKatG-lux) were studied by the bioluminescent test. Plant extracts from I. britannica and L. gmelinii in all used concentrations (0.5, 5.0, 50.0, and 500.0 μg/mL) had no genotoxic or oxidant activity. The extracts statistically significantly reduced the bioluminescence intensity of the pColD-lux, pKatG-lux, and pSoxS-lux sensors (p < 0.05) induced by 4-NQO and dioxidine, hydrogen peroxide, and paraquat, respectively. The activity of the extracts depended on their concentration; the greatest antigenotoxic and antioxidant effects were detected at a concentration of 500.0 μg/mL.     

Increase in Bacterial Resistance to Antibiotics after Cancer Therapy with Platinum-Based Drugs

The use of platinum-based anticancer drugs is limited by both their side effects and their effect on normal microflora’s metagenome. Drugs that possess mutagenic and genotoxic properties may cause mutations in microbial genomes that contribute to the emergence of resistance to antimicrobial preparations and the development of complications after chemotherapy. The effects of cisplatin and oxaliplatin on microorganisms were studied using bacterial biosensors—E. coli strains MG1655 pKatG-lux, which reacts to the generation of hydrogen peroxide; MG1655 pSoxS-lux, which reacts to the superoxide anion radical; and the MG1655 pColD-lux strain, which detects DNA damage. The biosensor tests demonstrated high levels of genotoxicity for both drugs and some differences in the spectrum of reactive oxygen species generated. Ascorbate reduced genotoxicity of cisplatin by 41%. Nonlethal doses of cisplatin induced a three- to sevenfold increase in the frequency of the mutations that confer the resistance of E. coli to rifampicin and ciprofloxacin. Ascorbate also reduced frequency of the mutations by 65%. Thus, the effect of these drugs was probably associated with the generation of reactive oxygen species and induction of SOS response. The risk of secondary antibiotic-resistant infections may be decreased by applying antioxidants and antimutagens. At the same time, these increases may also decrease the anti-tumoral action of these compounds.     

ATP limitation in a pyruvate formate lyase mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655 increases glycolytic flux to <Emphasis Type="SmallCaps">d</Emphasis>-lactate

A derivative strain of Escherichia coli MG1655 for d-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated “CL3.” Results show that the CL3 strain grew 44% slower than its parental strain under nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h. In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease in the ATP/ADP ratio causes an increase in the flux of glucose to lactate.     

Role of a flavonoid in the peroxide-dependent oxidation of glutathione catalysed by pea extracts

Crude pea extracts catalysed H₂O₂-dependent oxidation of glutathione but gel filtration through Sephadex G-25 abolished activity. Activity was restored by recombining the protein with a flavonoid [tentatively identified as kaempferol-3-(p-coumaroyltriglucoside)], isolated from peas. Protein fractions which supported peroxidase activity with pyrogallol as electron donor also supported H₂O₂-dependent oxidation of glutathione in the presence (but not in the absence) of the flavonoid with the concomitant consumption of O₂. In the absence of glutathione, active protein fractions also supported H₂O₂-dependent alteration of the spectral characteristics of the flavonoid. Some properties of these reactions were examined. It was concluded that these activities cannot be attributed to glutathione peroxidase and that a peroxidase belonging to EC class 1.11.1.7 is involved.     

Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance: New implications for osteonecrosis treatment?

Elevated hydrogen peroxide (H₂O₂) causes osteoblast dysfunction and apoptosis, serving as an important contributor to the development of osteonecrosis. Here we aimed to understand the role of AMP-activated protein kinase (AMPK) in the process. We observed a high level of AMPK activation in surgery isolated patients' osteonecrosis tissues. In cultured osteoblastoma MG63 cells, H₂O₂ stimulation induced significant AMPK activation, oxidative stress, cell death and apoptosis. Inhibition of AMPK by its inhibitor (compound C) or by shRNA-mediated knockdown dramatically enhanced H₂O₂-induced MG63 cell apoptosis, while over-expression of AMPK in HEK-293 cells alleviated H₂O₂-induced cell damage. These results confirmed that H₂O₂-activated AMPK is pro-cell survival. We observed that H₂O₂ induced protective autophagy in MG63 cells, and AMPK-dependent Ulk1 activation and mTORC1 (mTOR complex 1) inactivation might involve autophagy activation. Further, AMPK activation inhibited H₂O₂-induced oxidative stress, probably through inhibiting NADPH (nicotinamide adenine dinucleotide phosphate) depletion, since more NADPH depletion and oxidative stress were induced by H₂O₂ in AMPK deficient MG63 cells. Finally, we observed a significant AMPK activation in H₂O₂-treated primary cultured and transformed (MC3T3-E1) osteoblasts, and AMPK inhibitor compound C enhanced death by H₂O₂ in these cells. Based on these results, we concluded that H₂O₂-induced AMPK activation is pro-survival and anti-apoptosis in osteoblasts. Autophagy induction and NADPH maintenance are involved in AMPK-mediated pro-survival effects. AMPK might represent a novel molecular target for osteonecrosis treatment.     

Ferric Ion and Oxygen Reduction at the Surface of Protoplasts and Cells of Acer pseudoplatanus

This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O^?₂, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H₂O₂ that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H₂O₂ (catalase) or preventing H₂O₂ formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O^?₂, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H₂O₂ that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction).     

Vanillin Resistance Induced by BssS Overexpression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Overexpression of the BssS gene, a biofilm formation regulator, in planktonic Escherichia coli cells has been shown to confer the vanillin-resistant phenotype Van^r to the bacteria. The MG1655P_L-tac-bssS strain started growing in liquid aerated LB medium with 2 g/L vanillin after a lag phase of 17 ± 2 h, whereas the original MG1655 strain did not grow under these conditions. The role of aldehyde reductase YqhD, a vanillin- degrading enzyme, in Van^r phenotype formation has been assessed. However, the Van^r trait in the MG1655P_L-tac-bssS strain primarily depended on autoinducer-2 (AI-2), which formed in E. coli cells with an intact luxS gene. We supposed that BssS acts together with autoinducer-2 (which presumably accumulated during the prolonged lag phase) to induce vanillin resistance determined by changes in the expression of a range of genes.     

Kinetic simulation studies on the transient formation of the oxo‐iron(IV) porphyrin radical cation during the reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)‐porphyrin with hydrogen peroxide in aqueous solution

High‐valent oxo‐iron(IV) species are commonly proposed as the key intermediates in the catalytic mechanisms of iron enzymes. Water‐soluble iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin (Fe(III)TMPyP) has been used as a model of heme‐enzyme to catalyse the hydrogen peroxide (H₂O₂) oxidation of various organic compounds. However, the mechanism of the reaction of Fe(III)TMPyP with H₂O₂ has not been fully established. In this study, we have explored the kinetic simulation of the reaction of Fe(III)TMPyP with H₂O₂ and of the catalytic reactivity of FeTMPyP in the luminescent peroxidation of luminol. According to the mechanism that has been established in this work, Fe(III)TMPyP is oxidized by H₂O₂ to produce (TMPyP)^·+Fe(IV)=O (k1 = 4.5 × 10⁴/mol/L/s) as a precursor of TMPyPFe(IV)=O. The intermediate, (TMPyP)^·+Fe(IV)=O, represented nearly 2% of Fe(III)TMPyP but it does not accumulate in suf?cient concentration to be detected because its decay rate is too fast. Kinetic simulations showed that the proposed scheme is capable of reproducing the observed time courses of FeTMPyP in various oxidation states and the decay pro?les of the luminol chemiluminescence. It also shows that (TMPyP)^·+Fe(IV)=O is 100 times more reactive than TMPyPFe(IV)=O in most of the reactions. These two species are responsible for the initial sharp and the sustained luminol emissions, respectively. Copyright © 2003 John Wiley & Sons, Ltd.     

A comprehensive genome‐scale reconstruction of Escherichia coli metabolism—2011

The initial genome‐scale reconstruction of the metabolic network of Escherichia coli K‐12 MG1655 was assembled in 2000. It has been updated and periodically released since then based on new and curated genomic and biochemical knowledge. An update has now been built, named iJO1366, which accounts for 1366 genes, 2251 metabolic reactions, and 1136 unique metabolites. iJO1366 was (1) updated in part using a new experimental screen of 1075 gene knockout strains, illuminating cases where alternative pathways and isozymes are yet to be discovered, (2) compared with its predecessor and to experimental data sets to confirm that it continues to make accurate phenotypic predictions of growth on different substrates and for gene knockout strains, and (3) mapped to the genomes of all available sequenced E. coli strains, including pathogens, leading to the identification of hundreds of unannotated genes in these organisms. Like its predecessors, the iJO1366 reconstruction is expected to be widely deployed for studying the systems biology of E. coli and for metabolic engineering applications.     

Light generation with Fenton's reagent its relationship to granulocyte chemiluminescence

A simple chemical system consisting of FeSO₄ and H₂O₂ (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H₂O₂ was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH^.) and singlet oxygen (¹Δ_gO₂) quenchers suppressed chemiluminescence of the FeSO₄ + tryptophan + H₂O₂ system; and, deuterium oxide (²H₂O) enhanced chemiluminescence of both FeSO₄ reactions. These observations suggest that a radical chain reaction involving both OH^. and ¹Δ_gO₂ is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H₂O₂. Since iron and H₂O₂ are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.     

Intrinsic and inducible resistance to hydrogen peroxide in <Emphasis Type="Italic">Bifidobacterium</Emphasis> species

Interest in, and use of, bifidobacteria as a probiotic delivered in functional foods has increased dramatically in recent years. As a result of their anaerobic nature, oxidative stress can pose a major challenge to maintaining viability of bifidobacteria during functional food storage. To better understand the oxidative stress response in two industrially important bifidobacteria species, we examined the response of three strains of B. longum and three strains of B. animalis subsp. lactis to hydrogen peroxide (H₂O₂). Each strain was exposed to a range of H₂O₂ concentrations (0–10 mM) to evaluate and compare intrinsic resistance to H₂O₂. Next, strains were tested for the presence of an inducible oxidative stress response by exposure to a sublethal H₂O₂ concentration for 20 or 60 min followed by challenge at a lethal H₂O₂ concentration. Results showed B. longum subsp. infantis ATCC 15697 had the highest level of intrinsic H₂O₂ resistance of all strains tested and B. animalis subsp. lactis BL-04 had the highest resistance among B. lactis strains. Inducible H₂O₂ resistance was detected in four strains, B. longum NCC2705, B. longum D2957, B. lactis RH-1, and B. lactis BL-04. Other strains showed either no difference or increased sensitivity to H₂O₂ after induction treatments. These data indicate that intrinsic and inducible resistance to hydrogen peroxide is strain specific in B. longum and B. lactis and suggest that for some strains, sublethal H₂O₂ treatments might help increase cell resistance to oxidative damage during production and storage of probiotic-containing foods.     

Involvement of NADPH oxidase-mediated H<Subscript>2</Subscript>O<Subscript>2</Subscript> signaling in PB90-induced hypericin accumulation in <Emphasis Type="Italic">Hypericum perforatum</Emphasis> cells

PB90 is a novel protein elicitor isolated from Phytophthora boehmeriae. Here, we report that treatment of PB90 stimulates hypericin production and hydrogen peroxide (H₂O₂) generation in Hypericum perforatum L. cells and demonstrate that H₂O₂ is essential for PB90-induced hypericin production. To further study the source of PB90-triggered H₂O₂, we have investigated activities of plasma membrane NADPH oxidase in Hypericum perforatum L. cells subjected to PB90 treatment. It is revealed that treatment of the cells with PB90 significantly increases NADPH oxidase activity. NADPH oxidase inhibitors suppress not only the PB90-stimulated NADPH oxidase activity but also the PB90-triggered H₂O₂ generation and PB90-induced hypericin production, showing that NADPH oxidase is involved in PB90-triggered H₂O₂ generation and hypericin production. Moreover, the suppression of NADPH oxidase inhibitors on PB90-induced hypericin production can be reversed by H₂O₂, although H₂O₂ per se has no effects on hypericin production of the cells. Together, the data demonstrate that PB90 may induce hypericin production of H. perforatum cells through the NADPH oxidase-mediated H₂O₂ signaling pathway.