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1.
Pestic Biochem Physiol ; 170: 104687, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980055

RESUMO

Cytochrome P450 monooxygenases (P450s) are highly conserved multifunctional enzymes that play crucial roles in insecticide resistance development. In this study, the molecular mechanisms of P450s in acetamiprid resistance development to melon aphid, Aphis gossypii was investigated. Acetamiprid resistant (32.64-fold resistance) population (Ace-R) of A. gossypii was established by continuous selection with acetamiprid for 24 generations. Quantitative Real Time PCR was carried out to analyze the expression of P450 genes in both acetamiprid resistant (Ace-R) and susceptible (Ace-S) strains. Result showed that nine genes (CYP6CY14, CYP6DC1, CYP6CZ1, CYP6DD1, CYP6CY5, CYP6CY9, CYP6DA1, CYP6CY18, and CYP6CY16) of CYP3 clade, four genes (CYP302A1, CYP315A1, CYP301A1, and CYP314A1) of CYP2 clade, two genes (CYP4CK1, CYP4G51) of CYP4 clade and three genes (CYP306A1, CYP305E1, CYP307A1) of mitochondrial clade (Mito clad) were significantly up-regulated, in Ace-R compared to Ace-S strain. Whilst CYP4CJ2 gene from (CYP4 clade) was significantly down-regulated in Ace-R strain. Furthermore, RNA interference-mediated knockdown of CYP6CY14, CYP6DC1, and CYP6CZ1 genes significantly increased the sensitivity of Ace-R strain to acetamiprid. Taken together, this study showed that P450 genes especially CYP6CY14, CYP6DC1 and CYP6CZ1 are potentially involved in acetamiprid resistance development in A. gossypii. This study could be useful to understand the molecular basis of acetamiprid resistance mechanism in A. gossypii.


Assuntos
Afídeos/efeitos dos fármacos , Afídeos/genética , Cucurbitaceae , Inseticidas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Resistência a Inseticidas/efeitos dos fármacos , Resistência a Inseticidas/genética , Neonicotinoides
2.
Pestic Biochem Physiol ; 170: 104666, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980073

RESUMO

Despite the substantial progress achieved in the characterization of cytochrome P450 (CYP) -based resistance mechanisms in mosquitoes, a number of questions remain unanswered. These include: (i) the regulation and physiology of resistance conferring CYPs; (ii) the actual contribution of CYPs in resistance alone or in combination with other detoxification partners or other resistance mechanisms; (iii) the association between overexpression levels and allelic variation, with the catalytic activity and the intensity of resistance and (iv) the true value of molecular diagnostics targeting CYP markers, for driving decision making in the frame of Insecticide Resistance Management applications. Furthermore, the translation of CYP - based insecticide resistance research in mosquitoes into practical applications, is being developed, but it is not fully exploited, as yet. Examples include the production of high throughput platforms for screening the liability (stability) or inhibition potential of novel insecticidal leads and synergists (add-ons), as well as the exploration of the negative cross resistance concept (i.e. detoxification of certain insecticides, but activation of others pro-insecticides). The goal of this review is to critically summarise the current knowledge and the gaps of the CYP-based metabolic insecticide resistance in Anopheles and Aedes mosquito vectors. The progress and limitations of the protein and the reverse/forward genetic approaches, the understanding and importance of molecular and physiological aspects, as well as the current and future exploitation routes of CYP research are discussed.


Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Anopheles/efeitos dos fármacos , Anopheles/genética , Inseticidas/farmacologia , Piretrinas , Animais , Sistema Enzimático do Citocromo P-450/genética , Resistência a Inseticidas/efeitos dos fármacos , Resistência a Inseticidas/genética , Mosquitos Vetores/genética
3.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908132

RESUMO

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Biocatálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/ultraestrutura , Ensaios Enzimáticos , Metilação , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Estreptovaricina/biossíntese , Estreptovaricina/química , Estreptovaricina/metabolismo
4.
Yonsei Med J ; 61(8): 698-704, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32734733

RESUMO

PURPOSE: With changing fungal epidemiology and azole resistance in Aspergillus species, identifying fungal species and susceptibility patterns is crucial to the management of aspergillosis and mucormycosis. The objectives of this study were to evaluate performance of panfungal polymerase chain reaction (PCR) assays on formalin-fixed paraffin embedded (FFPE) samples in the identification of fungal species and in the detection of azole-resistance mutations in the Aspergillus fumigatus cyp51A gene at a South Korean hospital. MATERIALS AND METHODS: A total of 75 FFPE specimens with a histopathological diagnosis of aspergillosis or mucormycosis were identified during the 10-year study period (2006-2015). After deparaffinization and DNA extraction, panfungal PCR assays were conducted on FFPE samples for fungal species identification. The identified fungal species were compared with histopathological diagnosis. On samples identified as A. fumigatus, sequencing to identify frequent mutations in the cyp51A gene [tandem repeat 46 (TR46), L98H, and M220 alterations] that confer azole resistance was performed. RESULTS: Specific fungal DNA was identified in 31 (41.3%) FFPE samples, and of these, 16 samples of specific fungal DNA were in accord with a histopathological diagnosis of aspergillosis or mucormycosis; 15 samples had discordant histopathology and PCR results. No azole-mediating cyp51A gene mutation was noted among nine cases of aspergillosis. Moreover, no cyp51A mutations were identified among three cases with history of prior azole use. CONCLUSION: Panfungal PCR assay with FFPE samples may provide additional information of use to fungal species identification. No azole-resistance mediating mutations in the A. fumigatus cyp51A gene were identified among FFPE samples during study period.


Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica/genética , Hospitais , Mutação/genética , Idoso , Idoso de 80 Anos ou mais , Aspergillus fumigatus/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , República da Coreia
5.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1346-1355, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748592

RESUMO

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.


Assuntos
Comamonadaceae , Sistema Enzimático do Citocromo P-450 , Biologia Sintética , Catálise , Comamonadaceae/enzimologia , Comamonadaceae/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Expressão Gênica
6.
Plant Mol Biol ; 104(3): 327-337, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761540

RESUMO

KEY MESSAGE: Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.


Assuntos
Apiaceae/enzimologia , Apiaceae/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/metabolismo , Proteínas de Plantas/metabolismo , Apiaceae/genética , China , Cromatografia Líquida de Alta Pressão/métodos , Coenzima A Ligases/genética , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Furocumarinas/química , Furocumarinas/genética , Regulação da Expressão Gênica de Plantas , Cinética , Medicina Tradicional Chinesa , Fenilalanina Amônia-Liase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Transcriptoma
7.
Gene ; 758: 144961, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32693148

RESUMO

Ginkgo biloba L. is regarded as the most ancient living tree, and its kernel has been used as a traditional Chinese medicine for more than 2,000 years. The leaf extracts of this tree have been among the bestselling herbal remedies in Western countries since the last century. To understand the biosynthesis of the pharmacologically active ingredients in G. biloba, flavonoids and terpenoid trilactones (TTLs), we sequenced the transcriptomes of G. biloba leaves, kernels and testae with Iso-Seq and RNA-Seq technologies and obtained 152,524 clean consensus reads. When these reads were used to improve the annotation of the G. biloba genome, 4,856 novel genes, 25,583 new isoforms of previously annotated genes and 4,363 lncRNAs were discovered. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that genes involved in growth, regulation and response to stress were more likely to be regulated by alternative splicing (AS) or alternative polyadenylation (APA), which represent the two most important posttranscriptional regulation mechanisms. It was found that some of the characterized genes involved in the biosynthesis of flavonoids and TTLs were also possibly regulated by AS and APA. Using phylogenetic and gene expression pattern analyses, some candidate genes for the biosynthesis of flavonoids and TTLs were screened. After qRT-PCR validation, the final candidate genes for flavonoid biosynthesis included three UDP-glycosyltransferases and one MYB transcription factor, while the candidate genes for TTL biosynthesis included two cytochrome P450 and one WRKY transcription factor. Our study suggested that Iso-Seq may play an important role in improving genome annotation, elucidating AS and APA mechanisms and discovering candidate genes involved in the biosynthesis of some secondary metabolites.


Assuntos
Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas/genética , Ginkgo biloba/genética , Ginkgo biloba/metabolismo , Lactonas/metabolismo , Terpenos/metabolismo , Processamento Alternativo/genética , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica , Genoma de Planta/genética , Extratos Vegetais , Transcriptoma , Sequenciamento Completo do Genoma
8.
PLoS One ; 15(7): e0235746, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678853

RESUMO

Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.


Assuntos
Candida albicans/patogenicidade , Candidíase/microbiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo , Virulência , Sequência de Aminoácidos , Animais , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Esterol 14-Desmetilase/genética
9.
Chemosphere ; 254: 126802, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32660694

RESUMO

As the predominant predator of pests in rice fields, spiders have been exposed to cadmium (Cd) pollution for a long time. The livability of spiders during the overwintering period is closely related to population growth in spring, but the effects of Cd on spider's survival of cold hardness and the underlining mechanism remain unclear. In the present study, we found that some growth parameters (body length, width, mass and livability) in the wolf spider Pirata subpiraticus were altered distinctively under Cd stress. To investigate the effects of Cd toxicity on the spider at molecular levels, RNA-sequencing was performed on the spiderlings undergoing ambient temperature alterations. Transcriptome data showed that a total of 807 differentially expressed genes (DEGs) were yielded in the comparison. The obtained DEGs were mainly linked with metabolism-related process, including oxidoreductase activity and lipid transport, and 25 DEGs were associated with the reported cryoprotectants, including glycerol, arginine, cysteine, heat shock protein, glucose and mannose. Growth factors (insulin growth factor, platelet-derived growth factor and transforming growth factor) and cytochrome P450 encoding genes were dramatically expressed in the spider. Furthermore, transcriptional factors (TFs) family were characterized according to the transcriptomic profile, and ZBTB TFs were represented the most distinctive alterations in the characterized genes. Collectively, our study illustrated that Cd poses disadvantageous effects on the growth of P. subpiraticus at cold ambient temperature, and the spiders are capable of responding to the adverse Cd stress by expressing the genes involved in the metabolism of energy substances, cryoprotectants and immune-related components.


Assuntos
Cádmio/toxicidade , Resposta ao Choque Frio/efeitos dos fármacos , Aranhas/efeitos dos fármacos , Aranhas/fisiologia , Animais , Tamanho Corporal/efeitos dos fármacos , Tamanho Corporal/genética , Resposta ao Choque Frio/fisiologia , Sistema Enzimático do Citocromo P-450/genética , Poluentes Ambientais/toxicidade , Feminino , Perfilação da Expressão Gênica , Aranhas/genética , Fatores de Transcrição/genética , Transcriptoma
10.
Proc Natl Acad Sci U S A ; 117(26): 15262-15269, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541022

RESUMO

Thyroid hormone (TH) signaling plays an important role in the regulation of long-wavelength vision in vertebrates. In the retina, thyroid hormone receptor ß (thrb) is required for expression of long-wavelength-sensitive opsin (lws) in red cone photoreceptors, while in retinal pigment epithelium (RPE), TH regulates expression of a cytochrome P450 enzyme, cyp27c1, that converts vitamin A1 into vitamin A2 to produce a red-shifted chromophore. To better understand how TH controls these processes, we analyzed the phenotype of zebrafish with mutations in the three known TH nuclear receptor transcription factors (thraa, thrab, and thrb). We found that no single TH nuclear receptor is required for TH-mediated induction of cyp27c1 but that deletion of all three (thraa -/- ;thrab -/- ;thrb -/- ) completely abrogates its induction and the resulting conversion of A1- to A2-based retinoids. In the retina, loss of thrb resulted in an absence of red cones at both larval and adult stages without disruption of the underlying cone mosaic. RNA-sequencing analysis revealed significant down-regulation of only five genes in adult thrb -/- retina, of which three (lws1, lws2, and miR-726) occur in a single syntenic cluster. In the thrb -/- retina, retinal progenitors destined to become red cones were transfated into ultraviolet (UV) cones and horizontal cells. Taken together, our findings demonstrate cooperative regulation of cyp27c1 by TH receptors and a requirement for thrb in red cone fate determination. Thus, TH signaling coordinately regulates both spectral sensitivity and sensory plasticity.


Assuntos
Visão de Cores/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Opsinas/metabolismo , Receptores dos Hormônios Tireóideos/fisiologia , Percepção Visual/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Visão de Cores/genética , Sistema Enzimático do Citocromo P-450/genética , Deleção de Genes , Regulação da Expressão Gênica , Opsinas/genética , Células Fotorreceptoras Retinianas Cones , Raios Ultravioleta , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
11.
Proc Natl Acad Sci U S A ; 117(27): 15789-15798, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581129

RESUMO

Patients infected with influenza are at high risk of secondary bacterial infection, which is a major proximate cause of morbidity and mortality. We have shown that in mice, prior infection with influenza results in increased inflammation and mortality upon Staphylococcus aureus infection, recapitulating the human disease. Lipidomic profiling of the lungs of superinfected mice revealed an increase in CYP450 metabolites during lethal superinfection. These lipids are endogenous ligands for the nuclear receptor PPARα, and we demonstrate that Ppara -/- mice are less susceptible to superinfection than wild-type mice. PPARα is an inhibitor of NFκB activation, and transcriptional profiling of cells isolated by bronchoalveolar lavage confirmed that influenza infection inhibits NFκB, thereby dampening proinflammatory and prosurvival signals. Furthermore, network analysis indicated an increase in necrotic cell death in the lungs of superinfected mice compared to mice infected with S. aureus alone. Consistent with this, we observed reduced NFκB-mediated inflammation and cell survival signaling in cells isolated from the lungs of superinfected mice. The kinase RIPK3 is required to induce necrotic cell death and is strongly induced in cells isolated from the lungs of superinfected mice compared to mice infected with S. aureus alone. Genetic and pharmacological perturbations demonstrated that PPARα mediates RIPK3-dependent necroptosis and that this pathway plays a central role in mortality following superinfection. Thus, we have identified a molecular circuit in which infection with influenza induces CYP450 metabolites that activate PPARα, leading to increased necrotic cell death in the lung which correlates with the excess mortality observed in superinfection.


Assuntos
Inflamação/genética , Influenza Humana/genética , PPAR alfa/genética , Infecções Estafilocócicas/genética , Superinfecção/genética , Animais , Lavagem Broncoalveolar/métodos , Coinfecção/genética , Coinfecção/microbiologia , Coinfecção/mortalidade , Sistema Enzimático do Citocromo P-450/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Inflamação/microbiologia , Inflamação/mortalidade , Influenza Humana/microbiologia , Influenza Humana/mortalidade , Pulmão/microbiologia , Pulmão/patologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Camundongos Knockout , Necroptose/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/mortalidade , Superinfecção/mortalidade
12.
PLoS One ; 15(5): e0232626, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374762

RESUMO

The aim of this study is to determine the involvement of the flavonol-anthocyanin pathway on plant adaptation to biotic stress using the B.amyloliquefaciens QV15 to trigger blackberry metabolism and identify target genes to improve plant fitness and fruit quality. To achieve this goal, field-grown blackberries were root-inoculated with QV15 along its growth cycle. At fruiting, a transcriptomic analysis by RNA-Seq was performed on leaves and fruits of treated and non-treated field-grown blackberries after a sustained mildew outbreak; expression of the regulating and core genes of the Flavonol-Anthocyanin pathway were analysed by qPCR and metabolomic profiles by UHPLC/ESI-qTOF-MS; plant protection was found to be up to 88%. Overexpression of step-controlling genes in leaves and fruits, associated to lower concentration of flavonols and anthocyanins in QV15-treated plants, together with a higher protection suggest a phytoanticipin role for flavonols in blackberry; kempferol-3-O-rutinoside concentration was strikingly high. Overexpression of RuF3H (Flavonol-3-hidroxylase) suggests a pivotal role in the coordination of committing steps in this pathway, controlling carbon flux towards the different sinks. Furthermore, this C demand is supported by an activation of the photosynthetic machinery, and boosted by a coordinated control of ROS into a sub-lethal range, and associated to enhanced protection to biotic stress.


Assuntos
Adaptação Fisiológica , Antocianinas/metabolismo , Bacillus amyloliquefaciens/fisiologia , Sistema Enzimático do Citocromo P-450/fisiologia , Rubus/enzimologia , Rubus/microbiologia , Estresse Fisiológico , Sistema Enzimático do Citocromo P-450/genética , Frutas/enzimologia , Frutas/genética , Frutas/microbiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Rubus/genética
13.
Clin Drug Investig ; 40(7): 617-628, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32415468

RESUMO

BACKGROUND: Validated genomic biomarkers for oncological drugs are expanding to improve targeted therapies. Pharmacogenetics research focusing on the mechanisms underlying imatinib suboptimal response might help to explain the different treatment outcomes and drug safety profiles. OBJECTIVE: To investigate whether polymorphisms in genes encoding cytochrome P450 (CYP) enzymes and ABCB1 transporter affect imatinib pharmacokinetic parameters. METHODS: A prospective, multicenter, pharmacogenetic pilot study was performed in the context of two separate oral imatinib bioequivalence clinical trials, which included 26 healthy volunteers. DNA was extracted in order to analyze polymorphisms in genes CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and ABCB1. Imatinib plasma concentrations were measured by HPLC-MS/MS. Pharmacokinetic parameters were calculated by non-compartmental methods using WinNonlin software. RESULTS: Volunteers (n = 26; aged 24 ± 3 years; 69% male) presented regular pharmacokinetic imatinib data (concentration at 24 h, 436 ± 140 ng/mL and at 72 h, 40 ± 26 ng/mL; AUC0-72 32,868 ± 10,713 ng/mL⋅h; and Cmax 2074 ± 604 ng/mL). CYP2B6 516GT carriers showed a significant reduction of imatinib concentration at 24 h (23%, 391 ng/dL vs 511 ng/dL in 516GG carriers, p = 0.005) and elimination half-life (11%, 12.6 h vs 14.1 h in 516GG carriers, p = 0.041). Carriers for CYP3A4 (*22/*22, *1/*20 and *1/*22 variants) showed a reduced frequency of adverse events compared to *1/*1 carriers (0 vs 64%, p = 0.033). The other polymorphisms analyzed did not influence pharmacokinetics or drug toxicity. CONCLUSION: CYP2B6 G516T and CYP3A4 *20,*22 polymorphisms could influence imatinib plasma concentrations and safety profile, after single-dose administration to healthy subjects. This finding needs to be confirmed before it is implemented in clinical practice in oncological patients under treatment with imatinib.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Mesilato de Imatinib/farmacocinética , Polimorfismo Genético , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adulto , Feminino , Voluntários Saudáveis , Humanos , Mesilato de Imatinib/administração & dosagem , Masculino , Farmacogenética , Projetos Piloto , Estudos Prospectivos , Adulto Jovem
14.
Proc Biol Sci ; 287(1927): 20200838, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32453986

RESUMO

The putative synergistic action of target-site mutations and enhanced detoxification in pyrethroid resistance in insects has been hypothesized as a major evolutionary mechanism responsible for dramatic consequences in malaria incidence and crop production. Combining genetic transformation and CRISPR/Cas9 genome modification, we generated transgenic Drosophila lines expressing pyrethroid metabolizing P450 enzymes in a genetic background along with engineered mutations in the voltage-gated sodium channel (para) known to confer target-site resistance. Genotypes expressing the yellow fever mosquito Aedes aegypti Cyp9J28 while also bearing the paraV1016G mutation displayed substantially greater resistance ratio (RR) against deltamethrin than the product of each individual mechanism (RRcombined: 19.85 > RRCyp9J28: 1.77 × RRV1016G: 3.00). Genotypes expressing Brassicogethes aeneus pollen beetle Cyp6BQ23 and also bearing the paraL1014F (kdr) mutation, displayed an almost multiplicative RR (RRcombined: 75.19 ≥ RRCyp6BQ23: 5.74 × RRL1014F: 12.74). Reduced pyrethroid affinity at the target site, delaying saturation while simultaneously extending the duration of P450-driven detoxification, is proposed as a possible underlying mechanism. Combinations of target site and P450 resistance loci might be unfavourable in field populations in the absence of insecticide selection, as they exert some fitness disadvantage in development time and fecundity. These are major considerations from the insecticide resistance management viewpoint in both public health and agriculture.


Assuntos
Resistência a Inseticidas , Inseticidas/química , Aedes , Animais , Besouros , Sistema Enzimático do Citocromo P-450/genética , Mosquitos Vetores , Piretrinas
15.
PLoS One ; 15(5): e0231980, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357188

RESUMO

Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a ß-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen ß-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of ß-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in ß-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Transferases Intramoleculares/metabolismo , Ácido Oleanólico/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/análise , Plasmídeos/genética , Plasmídeos/metabolismo , Alinhamento de Sequência
16.
J Mycol Med ; 30(2): 100953, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32362445

RESUMO

OBJECTIVE: The present study was designed to discover novel biomarkers involved in voriconazole resistance in clinical isolates of Aspergillus flavus. MATERIALS AND METHODS: Two voriconazole non-wild-type and two voriconazole-wild-type A. flavus clinical isolates were selected to evaluate possible molecular mechanism involved in A. flavus resistance to voriconazole using the mutation assessment, Quantitative real- time PCR of cyp51A and cyp51C genes and complementary DNA- amplified fragment length polymorphism technique. RESULTS: No mutations were seen in the cyp51A and cyp51C genes in voriconazole non-wild-type isolates compared to wild- type and reference strains. Regarding to mRNA expression results, no changes were observed in expression fold of cyp51A and cyp51C mRNA expression level in first non- wild- type isolate compared to wild-type isolate. For second isolate cyp51C mRNA expression level was down regulated (5.6 fold). The set of genes including ABC fatty acid transporter XM- 002375835 and aldehydereductase XM- 002376518 and three unknown functional genes were identified. Based on results, the over-expression of AKR1 and ABC fatty acid transporter in the voriconazole non- wild- type isolates suggests these genes could represent a novel molecular marker linked to the voriconazole resistance in A. flavus. CONCLUSION: The results obtained in this study showed a novel finding as the authors identified AKR1 and ABC fatty acid transporter genes as possible voriconazole target genes in Iranian clinical isolates of A. flavus.


Assuntos
Aspergilose/microbiologia , Aspergillus flavus/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Voriconazol/uso terapêutico , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/genética , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/isolamento & purificação , Biomarcadores/análise , Sistema Enzimático do Citocromo P-450/genética , Análise Mutacional de DNA/métodos , Regulação Fúngica da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Técnicas de Tipagem Micológica , Mutação Puntual , Esterol 14-Desmetilase/genética
17.
J Biosci Bioeng ; 130(3): 265-271, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32423728

RESUMO

The efficient bioproduction of squalene-type triterpenoids (STs) has attracted considerable attention due to their significant biological activities. In a previous study, we constructed a recombinant Saccharomyces cerevisiae capable of producing three STs; 4,8-dihydroxy-22,23-oxidosqualene (ST-1), 8-hydroxy-2,3;22,23-squalene dioxide (ST-2), and 2,3;22,23-squalene dioxide (ST-3). Here, we first evaluated the effects of these STs on the growth of human non-small cell lung cancer (NSCLC) cells, and found that ST-3 exhibited the greatest potency compared to the other two STs. To further enhance the bioproduction of ST-3, we adopted a tunable system to balance the expression of the Ganoderma lucidum cytochrome P450 gene CYP505D13 in S. cerevisiae, which significantly improved the ST-3 production titer. The most effective strain produced 78.61 mg/L of ST-3 after 62 h fermentation, which was 6.43 times higher than that of our previous study. The present study demonstrated that ST-3 effectively inhibits the proliferation of NSCLC cells, and provides insight into its efficient bioproduction.


Assuntos
Biotecnologia , Sistema Enzimático do Citocromo P-450/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esqualeno/química , Esqualeno/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fermentação , Humanos , Neoplasias Pulmonares/patologia , Esqualeno/farmacologia
18.
Virology ; 546: 133-140, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452413

RESUMO

Auxin has profound effects on plant growth and development. In addition to participating in plant growth and development, the auxin signaling pathway is involved in plant defense against pathogens. In this study, we investigated the molecular mechanism by which helper-component protease (HCPro) encoded by the Tobacco vein banding mosaic virus (TVBMV) activates auxin biosynthesis genes (YUCs) and interferes with the auxin signaling pathway. Our results demonstrated that the viral suppressor HCPro decreased the DNA methylation of dispersed repeats (DRs) within the promoters of YUC1, YUC5 and YUC10 and transcriptional activated these YUC genes targeted by RNA-directed DNA methylation (RdDM), leading to an increase in auxin accumulation in plants. Furthermore, we found that the induction of these YUCs by HCPro was attenuated in ros1 mutant plants, suggesting that HCPro-mediated transcriptional activation of the genes was partly dependent on ROS1-mediated DNA demethylation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Ácidos Indolacéticos/metabolismo , Oxigenases/genética , Doenças das Plantas/virologia , Potyvirus/enzimologia , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Oxigenases/metabolismo , Doenças das Plantas/genética , Potyvirus/genética , Regiões Promotoras Genéticas
19.
Mutat Res ; 852: 503162, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265041

RESUMO

Diet is a major source of human exposure to polycyclic aromatic hydrocarbons (PAHs), of which benzo[a]pyrene (BaP) is the most commonly studied and measured. BaP has been considered to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes whose activity can be modulated by cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes. Previous studies showed that BaP-DNA adduct formation was greater in the livers of Hepatic Reductase Null (HRN) mice, in which POR is deleted specifically in hepatocytes, than in wild-type (WT) mice. In the present study we used human hepatoma HepG2 cells carrying a knockout (KO) in the POR gene as a human in vitro model that can mimic the HRN mouse model. Treatment to BaP for up to 48 h caused similar cytotoxicity in POR KO and WT HepG2 cells. However, levels of BaP activation (i.e. BaP-7,8-dihydrodiol formation) were higher in POR KO HepG2 cells than in WT HepG2 cells after 48 h. This also resulted in substantially higher BaP-DNA adduct formation in POR KO HepG2 cells indicating that BaP metabolism is delayed in POR KO HepG2 cells thereby prolonging the effective exposure of cells to unmetabolized BaP. As was seen in the HRN mouse model, these results suggest that cytochrome b5, another component of the mixed-function oxidase system, which can also serve as electron donor to CYP enzymes along with NADH:cytochrome b5 redutase, contributes to the bioactivation of BaP in POR KO HepG2 cells. Collectively, these findings indicate that CYPs play a more important role in BaP detoxication as opposed to activation.


Assuntos
Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/genética , Adutos de DNA/química , Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/deficiência , Adutos de DNA/agonistas , Adutos de DNA/metabolismo , Dano ao DNA , Relação Dose-Resposta a Droga , Expressão Gênica , Técnicas de Inativação de Genes , Células Hep G2 , Humanos
20.
Crit Rev Oncol Hematol ; 149: 102939, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32259776

RESUMO

A substantial proportion of cancer patients experience chemotherapy-induced nausea and vomiting (CINV) despite the use of antiemetic drugs. Prevalent genetic polymorphisms involved in antiemetic drug metabolism, drug transport and receptor pathways likely affect the effectiveness of antiemetics. Knowledge on which polymorphisms to integrate into individualised clinical care is needed. We did a systematic review evaluating the association between polymorphisms and effectiveness of antiemetics in cancer patients receiving moderately to highly emetogenic chemotherapy. Twenty studies n = 2331 evaluated eight polymorphisms in five candidate genes involved in 5-HT3 antagonist pathways. HTR3C C1214G increased the risk of acute chemotherapy-induced vomiting in the dominant model (odds ratio (OR) = 2.67, 95 % confidence interval (CI): 1.08-6.63). ABCB1 C3435T reduced the risk of acute CINV in the recessive model (OR = 0.60, 95 % CI: 0.44-0.81). Future studies should evaluate candidate genes that affect pharmacogenetics of other antiemetics beside 5-HT3 antagonists.


Assuntos
Antieméticos/uso terapêutico , Antineoplásicos/efeitos adversos , Sistema Enzimático do Citocromo P-450 , Náusea/prevenção & controle , Neoplasias/tratamento farmacológico , Farmacogenética , Receptores 5-HT3 de Serotonina , Antagonistas do Receptor 5-HT3 de Serotonina/uso terapêutico , Vômito/prevenção & controle , Antineoplásicos/uso terapêutico , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Humanos , Náusea/induzido quimicamente , Náusea/genética , Polimorfismo Genético/genética , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/genética , Vômito/induzido quimicamente , Vômito/genética
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