Cytochrome P450-dependent Monooxygenase Research Articles

Improved phenolic acid content and bioactivities of Salvia miltiorrhiza hairy roots by genetic manipulation of RAS and CYP98A14.

Phenolic acids from Salvia miltiorrhiza have been widely used in nutritious, health-promoting products with an increasing demand. In the current study, two biosynthetic genes RAS (rosmarinic acid synthase) and CYP98A14 (a cytochrome P450-dependent monooxygenase) were successfully introduced into S. miltiorrhiza hairy roots. Overexpression of RAS and CYP98A14 resulted in higher content of phenolic acids (up to over 3-fold) in transgenic lines compared to the control. Meanwhile, DPPH results revealed that engineered S. miltiorrhiza hairy roots had stronger antioxidant activities than the control. In addition, phenolic acid crude extracts of the engineered hairy root lines overexpressing RAS or CYP98A14 showed improved antibacterial activities compared to the control lines. Our work exhibits a useful strategy for enhancement of phenolic acid production and bioactivities of S. miltiorrhiza hairy roots by genetic manipulation of RAS and CYP98A14, and also provides a new resource material to obtain active phenolic acids for food and healthy products.

Biochemical Mechanisms, Cross-resistance and Stability of Resistance to Metaflumizone in Plutella xylostella.

The diamondback moth, Plutella xylostella (L.) is an important pest of cruciferous crops worldwide. It has developed resistance to many conventional and novel insecticide classes. Metaflumizone belongs to the new chemical class of semicarbazone insecticides. To delay the development of metaflumizone resistance in P. xylostella and to guide insecticide use in the field, the biochemical mechanisms, cross-resistance spectrum, and stability of resistance to metaflumizone were studied in a laboratory-selected resistant strain (metaflu-SEL). Synergism tests with the carboxylesterase inhibitor triphenyl phosphate (TPP), the glutathione S-transferase depletor diethyl maleate (DEM), and the P450 inhibitor piperonyl butoxide(PBO) had no obvious effect on metaflumizone in the metaflu-SEL strain and the susceptible strain (SS) of P. xylostella, with synergism ratios that ranged from 1.02 to 1.86. Biochemical studies revealed that the cytochrome P450-dependent monooxygenase increased only 1.13-fold in the metaflu-SEL strain compared with the UNSEL stain; meanwhile, carboxylesterase and glutathione S-transferase activity showed no difference. These results suggest that these detoxification enzymes may be not actively involved in metaflumizone resistance. Furthermore, the metaflu-SEL population showed a moderate level of cross-resistance to indoxacarb (11.63-fold), but only very low cross-resistance to spinosad (1.75-fold), spinetoram (3.52-fold), abamectin (2.81-fold), beta-cypermethrin (0.71-fold), diafenthiuron (0.79-fold), chlorantraniliprole (2.16-fold), BT (WG-001) (3.34-fold), chlorfenapyr (0.49-fold), and chlorfluazuron (0.97-fold). Moreover, metaflumizone resistance decreased from 1087.85- to 1.23-fold in the metaflu-SEL strain after 12 generations without exposure to metaflumizone. These results are useful for formulating insecticide resistance management strategies to control P. xylostella and to delay the development of metaflumizone resistance in the field.

Effects of frequently applied carbon monoxide releasing molecules (CORMs) in typical CO-sensitive model systems – A comparative in vitro study

Intracellular carbon monoxide (CO) is a gaseous signaling molecule and is generated enzymatically by heme oxygenases upon degradation of heme to billiverdin. Target structures for intracellular produced CO are heme proteins including cytochrome c oxidase of the respiratory chain, cytochrome P450-dependent monooxygenases, or myoglobin. For studies on CO signaling, CO-releasing molecules (CORMs) of different structure are available. Here, three frequently used CORMs (CORM-2, CORM-3 and CORM-401) were studied for their properties to provide CO in biological test systems and address susceptible heme proteins. CO release was investigated in the myoglobin binding assay and found to be rapid (<5 min) with CORM-2- and CORM-3, whereas CORM-401 continuously provided CO (>50 min). Storage stability of CORM stock solutions was also assessed with the myoglobin assay. Only CORM-401 stock solutions were stable over a period of 7 days. Incubation of CORMs with recombinant cytochrome P450 led to an inhibition of enzyme activity. However, only CORM-3 and CORM-401 proved to be suitable in this test system because controls with the inactivated CORM-2 (iCORM-2) also led to a loss of enzyme activity. The impact of CORMs on the respiratory chain was investigated with high resolution respirometry and extracellular flux technology. In the first approach interferences of CORM-2 and CORM-3 with oxygen measurement occurred, since a rapid depletion of oxygen was detected in the medium even when no cells were present. However, CORM-401 did not interfere with oxygen measurement and the expected inhibition of cellular respiration was observed. CORM-2 was not suitable for use in oxygen measurements with the extracellular flux technology and CORM-3 application did not show any effect in this system. However, CO-dependent inhibition of cellular respiration was observed with CORM-401. Based on the present experiments it is concluded, that CORM-401 produced most reliable CO-specific results for the modulation of typical CO targets. For studies on CO-dependent biological effects on intracellular heme groups, CORM-2 and CORM-3 were less suitable. Depending on the experimental setting, data achieved with these compounds should be evaluated with caution.

Enzymatic characterization of apple (Malus. x Domestica)

Secondary metabolites play a major role as quality factors in horticultural products by significantly contributing to plant health, optical attributes, sensory attributes and health beneficial effects for the consumer. Dihydrochalcones, a rare class of secondary metabolites, which is, however present in particularly high amounts in apple (Malus × domestica. In apple, phloridzin (phloretin 2’-O-glucoside) is prevalent representing more than 90% of the soluble phenolic compounds, but small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. For the systematic investigation, sufficiently comprehensive knowledge of the underlying pathway is yet lacking. Two types of enzymes could catalyze the reaction, polyphenol oxidases (PPOs) and/or cytochrome P450 dependent monooxygenases. To test a possible involvement of the F3‘H of apple in the formation of constitutive 3-hydroxydihydrochalcones, cDNA clones of the two types of F3‘Hs present in the apple was isolated and heterologously expressed in yeast. Despite showing high F3‘H enzyme activity with various flavonoid substrates, hydroxylation of dihydrochalcones was not observed at standard conditions, indicating that F3‘H is not part of the dihydrochalcone pathway.

SPR Analysis of Protein-Protein Interactions Involving Cytochromes P450 and Cytochrome b5 Integrated into Lipid Membrane

Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of modern protein interactomics. This study is a logical continuation of the cycle of our previous works aimed at the PPIs between components of the cytochrome P450-dependent monooxygenase system. Using an optical biosensor employing surface plasmon resonance (SPR biosensor), a comparative analysis characterizing kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 and cytochrome P450s was performed using two different protocols for protein immobilization: (1) covalent non-oriented immobilization on the carboxymethyl dextran chip and (2) non-covalent oriented immobilization in the lipid environment. In the case of the second protocol, PPIs were characterized by 2.5-fold higher affinity due to a decrease in rate dissociation constants values of the studied complexes.

Comparison of Various Aryl-Dithiolethiones and Aryl-Dithiolones As Hydrogen Sulfide Donors in the Presence of Rat Liver Microsomes.

It has been reported that microsomal metabolism of ADT (5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione, anetholedithiolethione, Sulfarlem) and ADO (5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one, anetholedithiolone) led to formation of H2S mainly derived from oxidations catalyzed by cytochrome P450-dependent monooxygenases and that ADO was a better H2S donor than ADT under these conditions. This article compares the H2S donor abilities of 18 dithiolethione and dithiolone analogs of ADT and ADO upon incubation with rat liver microsomes. It shows that, for all the studied compounds, maximal H2S formation was obtained after incubation with microsomes and NADPH and that this formation greatly decreased in the presence of N-benzylimidazole, a known inhibitor of cytochrome P450. This indicates that H2S formation from all the studied compounds requires, as previously observed in the case of ADT and ADO, oxidations catalyzed by cytochrome P450-dependent monooxygenases. Under these conditions, the studied dithiolones were almost always better H2S donors than the corresponding dithiolethiones. Interestingly, the best H2S yields (up to 75%) were observed in microsomal oxidation of ADO and its close analogs, pCl-Ph-DO and Ph-DO, in the presence of glutathione (GSH), whereas only small amounts of H2S were formed in microsomal incubations of those compounds with GSH but in the absence of NADPH. A possible mechanism for this effect of GSH is proposed on the basis of results obtained from reactions of GSH with 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one-1-sulfoxide, the ADO metabolite involved in H2S formation in microsomal oxidation of ADO. SIGNIFICANCE STATEMENT: A series of 18 dithiolethiones and dithiolones were compared for their ability to form hydrogen sulfide (H2S) in oxidations catalyzed by microsomal monooxygenases. The studied dithiolones were better H2S donors than the corresponding dithiolethiones, and the addition of glutathione to the incubations strongly increased H2S formation. A possible mechanism for this effect of GSH is proposed on the basis of results obtained from reactions of GSH with 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one-1-sulfoxide, a metabolite of the choleretic and sialologic drug Sulfarlem.

Aryl Hydrocarbon Receptor as a Target for Lycopene Preventing DEHP-Induced Spermatogenic Disorders.

Di(2-ethylhexyl)phthalate (DEHP) is widely used as a plasticizer to improve product flexibility and workability. Lycopene (LYC) is a natural compound and has promising preventive potentials, especially antireproductive toxicity, but the specific underlying mechanism is yet to be fully defined. Our study investigated the effect of LYC on DEHP-induced spermatogenesis disorders. Male ICR mice were treated with DEHP (500 or 1000 mg/kg BW/day) and/or LYC (5 mg/kg BW/day) for 28 days. Our results indicated that LYC could relieve the DEHP-induced injury of seminiferous tubules and spermatogenic cells, swelling of endoplasmic reticulum (ER), and an increase of mitochondria. LYC prevented increased levels of nuclear damage to DNA and the deformity rate and decreased values of sperm motility, number, and density. Moreover, LYC treatment decreased DEHP-induced nuclear accumulation of aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT), and the expressions of their downstream target genes such as cytochrome P450-dependent monooxygenases (CYP) 1A1, 1A2, and 1B1 were markedly reduced to normal in the LYC treatment group. Our study showed that LYC can prevent DEHP-induced spermatogenic disorders via an AHR/ARNT signaling system. This study provided new evidence of AHR as a target for LYC, which can prevent DEHP-induced toxicity.

Mechanisms of bacterial conversion and degradation of pharma pollutants from nonsteroidal anti-inflammatory drugs

Recently, there has been a steady increase in fundamental interest in studying the degree of bioavailability and toxic effects of pharmaceutical pollutants on natural microorganisms, which play the role of a primary response system to the xenobiotic load in the environment. Employing the bioresources of the Regional Specialised Collection of Alkanotrophic Microorganisms (IEGM, Large-Scale Research Facilities number 73559, WDCM # 768, http://www.iegmcol.ru), the ability of actinobacteria of the genus Rhodococcus to decompose complex aromatic compounds that constitute the non-steroidal anti-inflammatory drugs (NSAIDs) widely used for medical purposes was first established. Active biodegraders of diclofenac and ibuprofen, which dominate among NSAIDs detected in the natural environment and pose the greatest potential risk to hydrobiota and humans, were selected. The kinetics and main characteristics of the biodegradation process of the tested pharmaceutical pollutants were investigated depending on the physiological state and culture conditions of their biodegraders. Cytochrome P450-dependent monooxygenases were shown to participate in the initial oxidation of the pharma pollutants. The products of bacterial decomposition of the ecotoxicants were identified, the pathways of their biodegradation were characterized, and the mathematical models of the process of complete bacterial degradation of pharmaceutical pollutants in high concentrations were described. For the first time, evidence for the C-N bond breaking and aromatic ring opening in the diclofenac structure accompanied by the formation of metabolites harmless to living organisms was obtained. Potential bioactivity of individual metabolites of diclofenac and ibuprofen was evaluated. The most typical reactions of rhodococci exposed to NSAIDs were as follows: changes in the zeta potential, morphometric parameters and degree of hydrophobicity of bacterial cells, an increased content of total cellular lipids, and the formation of bacterial associates. The obtained results are considered to be mechanisms of rhodococci adaptation and their increased resistance to toxic effects of the pharmaceutical pollutants tested. The obtained fundamental data elucidate the environmental role of rhodococci in detoxification of pharma pollutants and underpin the implementation of innovative technical solutions for advanced pharmaceutical sewage treatment and the pharmaceutical waste disposal.

Abstract 2933: Application of a mouse model humanized for the major pathways of drug disposition in anticancer drug development and use

Abstract A vast number of targeted anticancer drugs are being developed in the pharmaceutical industry whose efficacy will only become fully realized through their combination with other established or novel anti-tumor agents. This is particularly the case because combination chemotherapy is a major approach being taken to overcome the rapid onset of drug resistance which current dosing regimens can cause. This has raised the conundrum of which of the large number of possible drug combinations, which may involve combinations of more than two drugs, have the greatest chance of success. It is not possible to test all the possible combinations by clinical trial alone so more informative preclinical models are badly needed. Nearly all targeted anti-tumor agents are substrates for the cytochrome P450-dependent monooxygenase system. The testing of novel drug regimens in mice is severely compromised by the major species differences in this enzyme system both in catalytic function, in the pattern of metabolite formation and the regulation by transcription factors such as CAR and PXR. In order to circumvent this problem we have created a mouse model where thirty four murine P450’s have been deleted from the mouse genome and substituted for the major enzymes involved in drug disposition in man ie CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP3A4 and CYP3A7. The mice have also been humanized for the transcription factors CAR and PXR. CYP3A4 and CYP2D6 are expressed off the human promoters. We report the validation the utility of this model by studying the in vivo metabolism and disposition of model drugs and the metabolism and disposition of the EGFR inhibitor, osimertinib and the BRAF inhibitor, dabrafenib. We show that the use of this model allows accurate prediction of clinically observed drug exposures, in the generation of human metabolites and drug/drug interactions. This model has therefore great potential for the development of combination therapies involving complex drug regimens and in the design of clinical trials targeted at overcoming drug resistance. Citation Format: Colin J. Henderson, Nico Scheer, Yury Kapelyukh, Aileen McLaren, Kenneth MacLeod, Anja Rode, De Lin, C Roland Wolf. Application of a mouse model humanized for the major pathways of drug disposition in anticancer drug development and use [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2933.

Mechanism of H2S Formation from the Metabolism of Anetholedithiolethione and Anetholedithiolone by Rat Liver Microsomes.

The drug anetholedithiolethione (ADT) and its analogs have been extensively used as H2S donors. However, the mechanism of H2S formation from ADT under biologic conditions remains almost completely unknown. This article shows that only small amounts of H2S are formed during incubation of ADT and of its metabolite anetholedithiolone (ADO) with rat liver cytosol or with rat liver microsomes (RLM) in the absence of NADPH, indicating that H2S formation under these conditions is of hydrolytic origin only to a minor extent. By contrast, much greater amounts of H2S are formed upon incubation of ADT and ADO with RLM in the presence of NADPH and dioxygen, with a concomitant formation of H2S and para-methoxy-acetophenone (pMA). Moreover, H2S and pMA formation under those conditions are greatly inhibited in the presence of N-benzyl-imidazole indicating the involvement of cytochrome P450-dependent monooxygenases. Mechanistic studies show the intermediate formation of the ADT-derived 1,2-dithiolium cation and of the ADO sulfoxide during microsomal metabolism of ADT and ADO, respectively. This article proposes the first detailed mechanisms for the formation of H2S from microsomal metabolism of ADT and ADO in agreement with those data and with previously published data on the metabolism of compounds involving a C=S bond. Finally, this article shows for the first time that ADO is a better H2S donor than ADT under those conditions. SIGNIFICANCE STATEMENT: Incubation of anetholedithiolethione (ADT) or its metabolite anetholedithiolone (ADO) in the presence of rat liver microsomes, NADPH, and O2 leads to H2S. This article shows for the first time that this H2S formation involves several steps catalyzed by microsomal monooxygenases and that ADO is a better H2S donor than ADT. We propose the first detailed mechanisms for the formation of H2S from the microsomal metabolism of ADT and ADO.

Biopesticide spinosad induces transcriptional alterations in genes associated with energy production in honey bees (Apis mellifera) at sublethal concentrations.

Bees experience substantial colony losses, which are often associated with pesticides. Besides synthetic insecticides biological compounds such as spinosad are used in agriculture and organic farming against insect pests. However, potential adverse effect at sublethal concentrations to pollinators are poorly known. Here we aim to determine potential adverse outcome pathways of spinosad and to identify molecular effects by investigating transcriptional alterations in the brain of honey bees. We experimentally exposed bees to three sublethal concentrations of 0.05, 0.5 and 5 ng spinosad/bee, and assessed transcriptional alterations of target genes. Additionally, we evaluated whether spinosad-induced transcriptional alterations were influenced by the time of the year. In April, alterations were most pronounced after 24 h exposure, while in June alterations occurred mostly after 48 h. In July, expressional alterations were often lower but the pattern was more similar to that in June than that in April. Down-regulation of genes encoding acetylcholine receptors, enzymes involved in oxidative phosphorylation (cox5a, ndufb7 and cox17), cytochrome P450 dependent monooxygenases (cyp9q1, cyp9q2 and cyp9q3) and insulin-like peptide-1 were among the most significant transcriptional alterations. This suggests adverse effects of spinosad to energy production and metabolism and thus negative consequences on foraging. Together, our study indicates that spinosad causes adverse effects at environmentally realistic concentrations, which may pose a risk to bee populations.

Carbon monoxide releasing molecule 401 (CORM-401) modulates phase I metabolism of xenobiotics

Next to its well-studied toxicity, carbon monoxide (CO) is recognized as a signalling molecule in various cellular processes. Thus, CO-releasing molecules (CORMs) are of considerable interest for basic research and drug development. Aim of the present study was to investigate if CO, released from CORMs, inhibits cytochrome P450-dependent monooxygenase (CYP) activity and modulates xenobiotic metabolism. CORM-401 was used as a model CO delivering compound; inactive CORM-401 (iCORM-401), unable to release CO, served as control compound. CO release from CORM-401, but not from iCORM-401, was validated using the cell free myoglobin assay. CO-dependent inhibition of CYP activity was shown by 7-ethoxyresorufin-O-deethylation (EROD) with recombinant CYP and HepG2 cells. Upon CORM-401 exposure EROD activity of recombinant CYP decreased concentration dependently, while iCORM-401 had no effect. Treatment with CORM-401 decreased EROD activity in HepG2 cells at concentrations higher than 50 μM CORM-401, while iCORM-401 showed no effect. At the given concentrations cell viability was not affected. Amitriptyline was selected as a model xenobiotic and formation of its metabolite nortriptyline by recombinant CYP was determined by HPLC. CORM-401 treatment inhibited the formation of nortriptyline whereas iCORM-401 treatment did not. Overall, we demonstrate CO-mediated inhibitory effects on CYP activity when applying CORMs. Since CORMs are currently under drug development, the findings emphasize the importance to take into account that this class of compounds may interfere with xenobiotic metabolism.

Elucidating diterpene biosynthesis in Setaria italica (foxtail millet) towards enhancing stress tolerance in food crops

My research focuses on elucidating the biosynthesis and biological activities of diterpenes in the model food crop Setaria italica (foxtail millet) with the goal of improving resistance against biotic and abiotic stresses in food crops. To increase global food production of agricultural crops by 70% for feeding an additional 2.3 billion people by 2050, knowledge of plant chemical defenses is necessary for limiting crop yield losses due to drought and disease. Plant terpenes are small molecule metabolites with critical roles in plant development (gibberellin phytohormones) and the defense against pests, pathogens and other environmental stresses such as drought and heavy metal stress. Key enzyme classes in forming the vast diversity of more than 70,000 plant terpenes are terpene synthases (TPS) and cytochrome P450‐dependent monooxygenases. TPS catalyze various cyclization and rearrangement reactions of prenyl‐diphosphates to produce the initial structural scaffolds, which are then further functionally modified (e.g. through hydroxylations) by P450 enzymes. Using our transcriptomics‐enabled gene discovery approach combined with plant‐based (Nicotiana benthamiana) and microbial (E. coli) co‐expression enzyme activity assays, and structure elucidation using GC‐MS and NMR analysis, we characterized the diterpene (20‐carbon terpene) synthase family of S. italica (SiDTPS) and one P450 (SiP450‐1) involved in C‐19 hydroxylation of diterpenes produced by SiDTPSs. S. italica may provide a unique insight into diterpene metabolism, since it is so far, the only species to contain all currently known diterpene precursors (copalyl diphosphate (CPP) of normal, ent‐, and syn‐ stereochemistry), whereas other major crops feature no more than two of the required enzymes. While (ent‐ and syn)‐CPP derived diterpenoids have been well characterized in rice (Oryza sativa) and maize (Zea mays) to confer quantitative pathogen defense, little is known about the role of (+)‐CPP‐derived diterpenes in crop stress resistance. Gene expression studies using qPCR show drought‐ and fungal elicitor (mycoprotein)‐inducible patterns of gene expression for select SiDTPSs and SiP450‐1 while diterpene products exhibit anti‐fungal activity in vitro against the major fungal pathogen, Fusarium verticilloides, suggesting a potential role in mediating fungal‐stress tolerance in planta. In collaboration with the DOE Joint Genome Institute, we will further work to develop LC‐MS protocols to detect the various identified diterpenoids from SiDTPS and SiP450‐1 diterpene enzyme products in planta and enable downstream genetic analysis of their biological activities.Support or Funding InformationDepartment of Plant Biology, UC Davis‐College of Biological SciencesJoint Genome Institute (DOE‐JGI), Grant CSP: DNA Synthesis #2568NIH‐NIGMS T32 GM007377, UC Davis Molecular and Cell Biology Training Grant ProgramDean's Mentorship Award 2017, UC Davis‐College of Biological ScienesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcriptomic analysis in Anemone flaccida rhizomes reveals ancillary pathway for triterpene saponins biosynthesis and differential responsiveness to phytohormones

Anemone flaccida Fr. Schmidt is a perennial medicinal herb that contains pentacyclic triterpenoid saponins as the major bioactive constituents. In China, the rhizomes are used as treatments for a variety of ailments including arthritis. However, yields of the saponins are low, and little is known about the plant's genetic background or phytohormonal responsiveness. Using one-quarter of the 454 pyrosequencing information from the Roche GS FLX Titanium platform, we performed a transcriptomic analysis to identify 157 genes putatively encoding 26 enzymes involved in the synthesis of the bioactive compounds. It was revealed that there are two biosynthetic pathways of triterpene saponins in A. flaccida. One pathway depends on β-amyrin synthase and is similar to that found in other plants. The second, subsidiary (“backburner”) pathway is catalyzed by camelliol C synthase and yields β-amyrin as minor byproduct. Both pathways used cytochrome P450-dependent monooxygenases (CYPs) and family 1 uridine diphosphate glycosyltransferases (UGTs) to modify the triterpenoid backbone. The expression of CYPs and UGTs were quite different in roots treated with the phytohormones methyl jasmonate, salicylic acid and indole-3-acetic acid. This study provides the first large-scale transcriptional dataset for the biosynthetic pathways of triterpene saponins and their phytohormonal responsiveness in the genus Anemone.

Duplicated flavonoid 3'-hydroxylase and flavonoid 3', 5'-hydroxylase genes in barley genome.

BackgroundAnthocyanin compounds playing multiple biological functions can be synthesized in different parts of barley (Hordeum vulgare L.) plant. The diversity of anthocyanin molecules is related with branching the pathway to alternative ways in which dihydroflavonols may be modified either with the help of flavonoid 3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H)—the cytochrome P450-dependent monooxygenases. The F3′H and F3′5′H gene families are among the least studied anthocyanin biosynthesis structural genes in barley. The aim of this study was to identify and characterise duplicated copies of the F3′H and F3′5′H genes in the barley genome.ResultsFour copies of the F3′5′H gene (on chromosomes 4HL, 6HL, 6HS and 7HS) and two copies of the F3′H gene (on chromosomes 1HL and 6HS) were identified in barley genome. These copies have either one or two introns. Amino acid sequences analysis demonstrated the presence of the flavonoid hydroxylase-featured conserved motifs in all copies of the F3′H and F3′5′H genes with the exception of F3′5′H-3 carrying a loss-of-function mutation in a conservative cytochrome P450 domain. It was shown that the divergence between F3′H and F3′5′H genes occurred 129 million years ago (MYA) before the emergence of monocot and dicot plant species. The F3′H copy approximately occurred 80 MYA; the appearance of F3′5′H copies occurred 8, 36 and 91 MYA. qRT-PCR analysis revealed the tissue-specific activity for some copies of the studied genes. The F3′H-1 gene was transcribed in aleurone layer, lemma and pericarp (with an increased level in the coloured pericarp), whereas the F3′H-2 gene was expressed in stems only. The F3′5′H-1 gene was expressed only in the aleurone layer, and in a coloured aleurone its expression was 30-fold higher. The transcriptional activity of F3′5′H-2 was detected in different tissues with significantly higher level in uncoloured genotype in contrast to coloured ones. The F3′5′H-3 gene expressed neither in stems nor in aleurone layer, lemma and pericarp. The F3′5′H-4 gene copy was weakly expressed in all tissues analysed.ConclusionF3′H and F3′5′H-coding genes involved in anthocyanin synthesis in H. vulgare were identified and characterised, from which the copies designated F3′H-1, F3′H-2, F3′5′H-1 and F3′5′H-2 demonstrated tissue-specific expression patterns. Information on these modulators of the anthocyanin biosynthesis pathway can be used in future for manipulation with synthesis of diverse anthocyanin compounds in different parts of barley plant. Finding both the copies with tissue-specific expression and a copy undergoing pseudogenization demonstrated rapid evolutionary events tightly related with functional specialization of the duplicated members of the cytochrome P450-dependent monooxygenases gene families.

Rapid multiplex gene expression assays for monitoring metabolic resistance in the major malaria vector Anopheles gambiae

BackgroundMetabolic resistance of the major malaria vector Anopheles gambiae (s.l.) to insecticides is operationally significant, particularly in combination with target site resistance. However, detection of metabolic resistance is not trivial and relies on laborious bioassays, unspecific biochemical methods, or sophisticated and expensive molecular approaches using transcriptomics.MethodsRapid one-step multiplex TaqMan-probe based RT-qPCR assays were developed and optimised to measure the expression levels of genes associated with metabolic insecticide resistance in An. gambiae (s.l.). Primers and probes were designed to target the mRNA of cytochrome P450-dependent monooxygenases CYP6P3, CYP6M2, CYP9K1, CYP6P4 and CYP6Z1, and the glutathione-S-transferase GSTE2. The novel assays were validated versus gold standard methods with a range of phenotyped mosquito specimens. The assays were also tested directly on lysates of RNAlater®-preserved mosquitoes without an RNA extraction step.ResultsThe novel assays are efficient (reaction efficiencies = 95–109%), sensitive (covering a > 10.0 Ct range with R2 values > 0.99), specific (TaqMan chemistry), reproducible (%CV = 4.46–12.07%), as well as readily expandable to capture additional loci as they evolve or to cover additional species. The assays were successfully validated in terms of expression levels against standard two-step singleplex qPCR assays (overall % difference = -17.6%, 95% CI = -38.7–3.43%) and microarrays, using laboratory strains and field-caught samples. The assays can also be applied directly on lysates of mosquito specimens, without RNA extraction or DNase treatment.ConclusionsThe novel multiplex assays for monitoring the levels of major detoxification genes and metabolic resistance in An. gambiae (s.l.) are simple to perform, robust and rapid. They may complement current diagnostic assays to provide evidence-based and operationally relevant information for insecticide resistance management.

SPR analysis of protein-protein interactions with P450 cytochromes and cytochrome b5 integrated into lipid membrane

Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of protein interactomics. This work is a logical continuation of the cycle of our previous works devoted to the study of PPIs among the components of cytochrome P450-dependent monooxygenase system. Using an optical biosensor of Surface Plasmon Resonance (SPR biosensor), a comparative analysis on the determination of kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 with cytochrome P450s was performed using two different protocols for protein immobilization: 1) covalent non-oriented one on to the carboxymethyl dextran chip type CM and 2) non-covalent oriented immobilization in the lipid environment on the chip type L1 with internal control of liposomes surface distribution. In the second protocol it was shown that the complex formation was characterized by 2.5 times higher affinity due to an decrease in rate dissociation constants. The appropriateness of using both experimental models is discussed.

Comparative expression of two detoxification genes by Callosobruchus maculatus in response to dichlorvos and Lippia adoensis essential oil treatments

The cowpea beetle, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae), is a field-to-store pest, which can cause up to 80% damage of cowpea grains within 3 months of storage. The control approach consisting of application of synthetic pesticides has become challenging following the increased resistance and toxicity to non-target organisms and the environment. Here, we hypothesized that Lippia adoensis essential oil (EO) (plant-based insecticide) can repress cytochrome P450-dependent mono-oxygenase and glutathione-S-transferase (GST) genes and suppress C. maculatus resistance to dichlorvos (O,O-dimethyl-O-2,2-dichlorovinylphosphate or DDVP). The methods consisted of separately exposing C. maculatus adults to cowpea seeds treated with DDVP and L. adoensis EO. Their physiological and molecular responses were monitored for five generations. Adult mortality of DDVP-treated beetles significantly decreased across generations and negatively correlated with the reproduction parameters (increase in fecundity and adult emergence) and seed damage. Similarly, the decrease in adult susceptibility corresponded with the increase in the expression levels of cytochrome P450 and GST genes (overexpression of genes). However, the adult susceptibility to EO treatments remained consistent across generations and correlated with the down-regulation of targeted genes from the third generation (F3). These results support our hypotheses and provide a probable molecular basis of resistance to DDVP and susceptibility to L. adoensis EO in C. maculatus. Therefore, L. adoensis EO represents an alternative insecticide that could be employed to enhance the vulnerability of this pest.

Hypolipidemic and Hypoglycemic Effects of Hydroalcoholic Extract of Solanum nigrum Linn. in CCl4-induced Hepatotoxicity in Mice

Carbon tetrachloride (CCl4) toxicity is the model commonly exploited to produce hepatic damage. This model is used to screen drugs with hepatoprotective activity on various experimental animals and to validate their liver protecting property. Carbon tetrachloride gets accumulated in hepatic parenchymal cells and is metabolically activated by cytochrome P450-dependent monooxygenases to generate free radicals which covalently bind with tissue macromolecules like carbohydrates and proteins causing disturbances in cellular homeostasis. This may lead to the initiation of lipid peroxidation a sequence of chain reactions in cellular membranes which ultimately may result in steatosis. The present study was an attempt to study the anti-hepatotoxic, hypolipidemic and hypoglycaemic effect of S. nigrum against CCl4 - induced hepatotoxicity in female mice. The hypolipidemic, hypoglycaemic and hepatoprotective activity of hydroalcoholic extract of S. nigrum was evaluated by various biochemical parameters and by histopathological examination. Carbon tetrachloride administration caused a significant increase in liver total lipids, triglyceride (TG), cholesterol and free fatty acid content. Similarly serum low density lipoproteins (LDL-C), very low density lipoprotein levels (VLDL-C) and bilirubin were elevated after toxin administration, while serum high density lipoproteins (HDL-C) was lowered as compared to vehicle control. It was observed that oral administration of the toxin caused a significant increase in blood glucose level, while a significant decrease was observed in the glycogen and protein content of the liver and albumin content of serum as compared to vehicle control. Oral administration of S. nigrum effectively mitigated the changes induced by CCl4 in a dose - dependent manner. Our results indicated S. nigrum exerts hepatoprotective property by stabilizing tissue macromolecules resulting in maintenance of cellular hemeostasis.

Field-evolved resistance to chlorantraniliprole in the tomato pinworm Tuta absoluta: inheritance, cross-resistance profile, and metabolism

The tomato pinworm Tuta absoluta is currently the most important pest of tomatoes worldwide. Diamides are one of the few effective classes of insecticides in use for its management, but recent cases of very high resistance to these insecticides in field populations of this invasive pest species have been reported in Brazil and Europe. Herein, the resistance of T. absoluta to chlorantraniliprole was genetically and biochemically characterized. Concentration–mortality curves were estimated for parental (GBN-Sus and AMD-Sel), F1, and backcross generations through the leaf-dipping method, which was also used in further bioassay-based experiments. Inheritance studies indicated that chlorantraniliprole resistance is monogenic, completely recessive, and autosomal. Dominance of chlorantraniliprole resistance was concentration dependent shifting to recessive as the insecticide concentration increased. In addition, concentrations above 0.4 mg chlorantraniliprole/L made resistance completely recessive. Esterases, glutathione S-transferases, and cytochrome P450-dependent monooxygenases are apparently not involved in the chlorantraniliprole resistance. Furthermore, the cross-resistance spectrum was restricted to the diamide insecticides flubendiamide and cyantraniliprole, suggesting that altered target site is the likely mechanism involved, in contrast with enhanced detoxification. Therefore, the interruption of chlorantraniliprole use against the tomato pinworm would be a useful management initiative for diamide resistance management for already resistant populations. Diamide use against this species in newly invaded areas should be carefully monitored to ascertain that diamide-resistant insects were not introduced and also to prevent rapid selection for diamide resistance and its potential spread to other areas.