Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.015
Filtrar
1.
J Neuroimmunol ; 361: 577724, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34610503

RESUMO

Prostaglandin E2 (PGE2) plays pivotal roles in controlling microglial activation with the EP2 receptor, a PGE2 receptor subtype. Activated microglia are often reported to increase cyclooxygenase (COX)-2 expression, followed by PGE2 production, but it is unclear whether extracellular PGE2 is involved in microglial PGE2 synthesis. In the present study, we report that PGE2 increases COX-2 protein in microglia. In a culture system, PGE2 at 10-6 M for 3 h increased COX-2 and microsomal PGE synthase (mPGES)-1 mRNA levels, and reduced mPGES-2, but did not affect COX-1 or cytosolic PGE synthase (cPGES) in microglia. PGE2 at 10-6 M for 3 h also increased the COX-2 protein level, but did not affect COX-1, mPGES-1, mPGES-2, or cPGES. An EP2 agonist, ONO-AE1-259-01, also increased COX-2 and mPGES-1 mRNA levels, and reduced mPGES-2, but did not affect COX-1 or cPGES, whereas an EP1 agonist, ONO-DI-004, an EP3 agonist, ONO-AE-248, and an EP4 agonist, ONO-AE1-329, had no effect. Similar to PGE2, ONO-AE1-259-01 increased the COX-2 protein level, but did not affect COX-1, mPGES-1, mPGES-2, or cPGES. In addition, the effects of PGE2 were inhibited by an EP2 antagonist, PF-04418948, but not by an EP1 antagonist, ONO-8713, an EP3 antagonist, ONO-AE3-240, or an EP4 antagonist, ONO-AE3-208, at 10-6 M. On the other hand, lipopolysaccharide (LPS) increased PGE2 production, but the LPS-induced PGE2 production was not affected by ONO-8713, PF-04418948, ONO-AE3-240, or ONO-AE3-208. These results indicate that PGE2 increases COX-2 protein in microglia through the EP2 receptor supporting the idea that extracellular PGE2 has a triggering aspect for microglial activation.


Assuntos
Ciclo-Oxigenase 2/biossíntese , Dinoprostona/farmacologia , Microglia/efeitos dos fármacos , Animais , Azetidinas/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Dinoprostona/análogos & derivados , Dinoprostona/biossíntese , Indução Enzimática/efeitos dos fármacos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Éteres Metílicos/farmacologia , Microglia/enzimologia , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Prostaglandina-E Sintases/biossíntese , Prostaglandina-E Sintases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores
2.
Future Oncol ; 17(35): 4925-4946, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34672683

RESUMO

Aim: Epoxide hydrolase is involved in oxidative defenses and is responsible for the activation of carcinogens. The relationship between EPHX1 polymorphisms (Tyr113His and His139Arg) and overall survival (OS) and lung cancer (LC) risk was investigated. Methods: The study comprised 550 cases and 550 controls. Genotyping and statistical analysis were applied. Results: The variant genotypes of EPHX1 polymorphisms exhibited no association with LC risk. The Tyr113His polymorphism exhibited twofold increased odds of lymph node invasion (p = 0.04). The Tyr/His genotype is a risk factor for smokers. Subjects carrying the combined genotype for His139Arg showed better median survival time (MST) and the heterozygous genotype revealed better MST in the case of small-cell lung cancer (SCLC; 11.30 vs 6.73 months; log-rank test: p = 0.02). The heterozygous genotype (His139Arg) had longer MST in patients receiving cisplatin/carboplatin and irinotecan (11.30 vs 7.23; log-rank test: p = 0.007) Conclusion: The Tyr113His polymorphism is associated with LC risk in smokers and is a potential prognostic factor for OS in patients with SCLC after irinotecan.


Lay abstract Microsomal epoxide hydrolase (mEH) is an enzyme that plays a defensive role against chemicals. In this study, the relationship between the variation of the epoxide hydrolase and the risk of lung cancer was investigated and its role in the survival of patients with lung cancer was evaluated. The study comprised 550 cases and 550 controls. Genotyping was carried out using molecular biology tools and was followed by statistical analysis. The variant genotype of the EPHX1 gene was not associated with the risk of lung cancer, even based on histology. The variant form of the EPHX1 gene was found to be a risk factor for smokers. The Tyr/His genotype was associated with the risk of lung cancer in male subjects. Patients carrying the variant form of the EPHX1 gene (His139Arg) experienced better survival. The heterozygous genotype of the EPHX1Tyr113His gene was related to longer survival time in patients who received cisplatin/carboplatin along with irinotecan. The EPHX1 Tyr113His polymorphism is associated with LC risk in smokers and is a potential prognostic factor for OS of patients with small-cell lung cancer (SCLC) after irinotecan therapy and might increase the likelihood of lymph node metastasis; EPHX1His139Arg exhibited better survival, especially in patients with SCLC.


Assuntos
Suscetibilidade a Doenças , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/mortalidade , Microssomos/enzimologia , Polimorfismo Genético , Fumar Tabaco/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Substituição de Aminoácidos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Variação Genética , Genótipo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Razão de Chances , Platina/administração & dosagem , Prognóstico
3.
Basic Clin Pharmacol Toxicol ; 129(6): 427-436, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34396687

RESUMO

Because several steroid hormones are metabolized to their respective 6ß-hydroxy forms by CYP3A4 and CYP3A5, these isoenzymes have been assumed to metabolize the immunosuppressive drug prednisolone, with conflicting results in the literature with respect to their relative importance. A direct study of the metabolism of prednisolone by microsomal CYP3A4 and CYP3A5 is missing. The aim of this in vitro study was to investigate the relative importance of recombinant CYP3A4 and recombinant CYP3A5 in the metabolism of prednisolone and to compare the extent of formation of 6ß-OH-prednisolone by the two enzymes. Through in vitro incubations using rCYP3A4 and rCYP3A5 enzymes, intrinsic clearance (CLint ) of prednisolone was determined by the substrate depletion approach. Formation of the metabolite 6ß-OH-prednisolone by rCYP3A4 and rCYP3A5, respectively, was compared. Prednisolone concentrations were measured, and its metabolite 6ß-OH-prednisolone was identified using a HPLC-MS/MS in-house method. CLint for prednisolone by rCYP3A5 was less than 26% relative to rCYP3A4. Formation of 6ß-OH-prednisolone by rCYP3A5 was less than 11% relative to rCYP3A4. The study indicates that 6ß-hydroxylation of prednisolone assessed in vitro in recombinant CYP enzymes depends on rCYP3A4 rather than rCYP3A5 and that CYP3A5 may be responsible for the formation of other prednisolone metabolite(s) in addition to 6ß-OH-prednisolone.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Glucocorticoides/metabolismo , Prednisolona/análogos & derivados , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Hidroxilação , Insetos , Microssomos/enzimologia , Prednisolona/metabolismo , Espectrometria de Massas em Tandem
4.
Sci Rep ; 11(1): 16580, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400719

RESUMO

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.


Assuntos
Proteínas de Membrana/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Ativação Enzimática/efeitos dos fármacos , Células Gigantes/enzimologia , Humanos , Células-Tronco Pluripotentes Induzidas/enzimologia , Microssomos/enzimologia , Simulação de Dinâmica Molecular , Estrutura Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Fosfatidilcolinas , Domínios Proteicos/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Bibliotecas de Moléculas Pequenas/efeitos adversos , Bibliotecas de Moléculas Pequenas/farmacologia , Suínos , Água
5.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360902

RESUMO

Acyl-CoA:lysophosphatidylethanolamine acyltransferases (LPEATs) are known as enzymes utilizing acyl-CoAs and lysophospholipids to produce phosphatidylethanolamine. Recently, it has been discovered that they are also involved in the growth regulation of Arabidopsis thaliana. In our study we investigated expression of each Camelina sativa LPEAT isoform and their behavior in response to temperature changes. In order to conduct a more extensive biochemical evaluation we focused both on LPEAT enzymes present in microsomal fractions from C. sativa plant tissues, and on cloned CsLPEAT isoforms expressed in yeast system. Phylogenetic analyses revealed that CsLPEAT1c and CsLPEAT2c originated from Camelina hispida, whereas other isoforms originated from Camelina neglecta. The expression ratio of all CsLPEAT1 isoforms to all CsLPEAT2 isoforms was higher in seeds than in other tissues. The isoforms also displayed divergent substrate specificities in utilization of LPE; CsLPEAT1 preferred 18:1-LPE, whereas CsLPEAT2 preferred 18:2-LPE. Unlike CsLPEAT1, CsLPEAT2 isoforms were specific towards very-long-chain fatty acids. Above all, we discovered that temperature strongly regulates LPEATs activity and substrate specificity towards different acyl donors, making LPEATs sort of a sensor of external thermal changes. We observed the presented findings not only for LPEAT activity in plant-derived microsomal fractions, but also for yeast-expressed individual CsLPEAT isoforms.


Assuntos
Aciltransferases/metabolismo , Camellia/enzimologia , Camellia/genética , Fosfatidiletanolaminas/metabolismo , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Temperatura , Acil Coenzima A/metabolismo , Aciltransferases/genética , Camellia/classificação , Camellia/crescimento & desenvolvimento , Resposta ao Choque Frio , DNA de Plantas/genética , Ativação Enzimática , Resposta ao Choque Térmico , Isoenzimas/genética , Microssomos/enzimologia , Filogenia , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Especificidade por Substrato
6.
Drug Metab Dispos ; 49(9): 718-728, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34135089

RESUMO

For drug development, species differences in drug-metabolism reactions present obstacles for predicting pharmacokinetics in humans. We characterized the species differences in hydrolases among humans and mice, rats, dogs, and cynomolgus monkeys. In this study, to expand the series of such studies, we attempted to characterize marmoset hydrolases. We measured hydrolase activities for 24 compounds using marmoset liver and intestinal microsomes, as well as recombinant marmoset carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC). The contributions of CES1, CES2, and AADAC to hydrolysis in marmoset liver microsomes were estimated by correcting the activities by using the ratios of hydrolase protein levels in the liver microsomes and those in recombinant systems. For six out of eight human CES1 substrates, the activities in marmoset liver microsomes were lower than those in human liver microsomes. For two human CES2 substrates and three out of seven human AADAC substrates, the activities in marmoset liver microsomes were higher than those in human liver microsomes. Notably, among the three rifamycins, only rifabutin was hydrolyzed by marmoset tissue microsomes and recombinant AADAC. The activities for all substrates in marmoset intestinal microsomes tended to be lower than those in liver microsomes, which suggests that the first-pass effects of the CES and AADAC substrates are due to hepatic hydrolysis. In most cases, the sums of the values of the contributions of CES1, CES2, and AADAC were below 100%, which indicated the involvement of other hydrolases in marmosets. In conclusion, we clarified the substrate preferences of hydrolases in marmosets. SIGNIFICANCE STATEMENT: This study confirmed that there are large differences in hydrolase activities between humans and marmosets by characterizing marmoset hydrolase activities for compounds that are substrates of human CES1, CES2, or arylacetamide deacetylase. The data obtained in this study may be useful for considering whether marmosets are appropriate for examining the pharmacokinetics and efficacies of new chemical entities in preclinical studies.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Hidrolases , Intestino Delgado/enzimologia , Fígado/enzimologia , Microssomos/enzimologia , Rifamicinas/farmacocinética , Animais , Callithrix , Carboxilesterase/metabolismo , Desenvolvimento de Medicamentos/métodos , Ativação Enzimática/fisiologia , Ensaios Enzimáticos/métodos , Humanos , Hidrolases/classificação , Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Especificidade por Substrato
7.
Int J Mol Sci ; 22(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066758

RESUMO

Epoxy-fatty acids (EpFAs) are endogenous lipid mediators that have a large breadth of biological activities, including the regulation of blood pressure, inflammation, angiogenesis, and pain perception. For the past 20 years, soluble epoxide hydrolase (sEH) has been recognized as the primary enzyme for degrading EpFAs in vivo. The sEH converts EpFAs to the generally less biologically active 1,2-diols, which are quickly eliminated from the body. Thus, inhibitors of sEH are being developed as potential drug therapeutics for various diseases including neuropathic pain. Recent findings suggest that other epoxide hydrolases (EHs) such as microsomal epoxide hydrolase (mEH) and epoxide hydrolase-3 (EH3) can contribute significantly to the in vivo metabolism of EpFAs. In this study, we used two complementary approaches to probe the relative importance of sEH, mEH, and EH3 in 15 human tissue extracts: hydrolysis of 14,15-EET and 13,14-EDP using selective inhibitors and protein quantification. The sEH hydrolyzed the majority of EpFAs in all of the tissues investigated, mEH hydrolyzed a significant portion of EpFAs in several tissues, whereas no significant role in EpFAs metabolism was observed for EH3. Our findings indicate that residual mEH activity could limit the therapeutic efficacy of sEH inhibition in certain organs.


Assuntos
Epóxido Hidrolases/metabolismo , Ácidos Graxos/metabolismo , Microssomos/enzimologia , Especificidade de Órgãos , Epóxido Hidrolases/antagonistas & inibidores , Humanos , Hidrólise , Cinética , Proteínas Recombinantes/metabolismo , Solubilidade , Especificidade por Substrato , Extratos de Tecidos
8.
Future Med Chem ; 13(13): 1091-1103, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34080888

RESUMO

Aim: This study investigated our Enzymelinks, COX-2-10aa-mPGES-1 and COX-2-10aa-PGIS, as cellular cross-screening targets for quick identification of lead compounds to inhibit inflammatory PGE2 biosynthesis while maintaining prostacyclin synthesis. Methods: We integrated virtual and wet cross-screening using Enzymelinks to rapidly identify lead compounds from a large compound library. Results: From 380,000 compounds virtually cross-screened with the Enzymelinks, 1576 compounds were identified and used for wet cross-screening using HEK293 cells that overexpressed individual Enzymelinks as targets. The top 15 lead compounds that inhibited mPGES-1 activity were identified. The top compound that specifically inhibited inflammatory PGE2 biosynthesis alone without affecting COX-2 coupled to PGI2 synthase (PGIS) for PGI2 biosynthesis was obtained. Conclusion: Enzymelink technology could advance cyclooxygenase pathway-targeted drug discovery to a significant degree.


Assuntos
Derivados de Benzeno/farmacologia , Ciclo-Oxigenase 1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredutases Intramoleculares/metabolismo , Engenharia de Proteínas , Derivados de Benzeno/química , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia
9.
J Ethnopharmacol ; 271: 113914, 2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-33571617

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Shengmai San (SMS) has been commonly used as a traditional Chinese medicine for the treatment of cardiovascular disorders, of which drug interactions need to be assessed for the safety concern. There is little evidence for the alterations of hepatic and intestinal drug-metabolizing enzymes after repeated SMS treatments to assess drug interactions. AIM OF THE STUDY: The studies aim to illustrate the effects of repeated treatments with SMS on cytochrome P450s (CYPs), reduced nicotinamide adenine dinucleotide (phosphate)-quinone oxidoreductase (NQO), uridine diphosphate-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) using in vivo rat model. MATERIALS AND METHODS: The SMS was prepared using Schisandrae Fructus, Ginseng Radix, and Ophiopogonis Radix (OR) (1:2:2). Chromatographic analyses of decoctions were performed using ultra-performance liquid chromatography (UPLC) and LC-mass spectrometry. Sprague-Dawley rats were orally treated with the SMS and its component herbal decoctions for 2 or 3 weeks. Hepatic and intestinal enzyme activities were determined. CYP3A expression and the kinetics of intestinal nifedipine oxidation (NFO, a CYP3A marker reaction) were determined. RESULTS: Schisandrol A, schisandrin B, ginsenoside Rb1 and ophiopogonin D were identified in SMS. SMS selectively suppressed intestinal, but not hepatic, NFO activity in a dose- and time-dependent manner. Hepatic and intestinal UGT, NQO and GST activities were not affected. A 3-week SMS treatment decreased the maximal velocity of intestinal NFO by 50%, while the CYP3A protein level remained unchanged. Among SMS component herbs, the decoction of OR decreased intestinal NFO activity. CONCLUSIONS: These findings demonstrate that 3-week treatment with SMS and OR suppress intestinal, but not hepatic CYP3A function. It suggested that the potential interactions of SMS with CYP 3A drug substrates should be noticed, especially the drugs whose bioavailability depends heavily on intestinal CYP3A.


Assuntos
Inibidores do Citocromo P-450 CYP3A/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Intestinos/enzimologia , Fígado/enzimologia , Animais , Biomarcadores/sangue , Ciclo-Octanos/análise , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/análise , Inibidores do Citocromo P-450 CYP3A/uso terapêutico , Combinação de Medicamentos , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/uso terapêutico , Ginsenosídeos/análise , Glucuronosiltransferase/metabolismo , Glutationa Transferase/metabolismo , Interações Ervas-Drogas , Intestinos/efeitos dos fármacos , Lignanas/análise , Fígado/efeitos dos fármacos , Masculino , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Nifedipino/metabolismo , Oxirredução/efeitos dos fármacos , Compostos Policíclicos/análise , Ratos Sprague-Dawley , Saponinas/química , Espirostanos/química
10.
Arch Biochem Biophys ; 701: 108791, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33592181

RESUMO

Human cytochrome P450 enzymes (CYPs or P450s) are known to be reduced by their electron transfer partners in the absence of substrate and in turn to reduce other acceptor molecules such as molecular oxygen, thereby creating superoxide anions (O2-•). This process is known as futile cycling. Using our previously established fission yeast expression system we have monitored cells expressing each one of the 50 human microsomal CYPs in the absence of substrate for oxidation of dihydroethidium in living cells by flow cytometry. It was found that 38 of these display a statistically significant increase in O2-• production. More specifically, cells expressing some CYPs were found to be intermediate strength O2-• producers, which means that their effect was comparable to that of treatment with 3 mM H2O2. Cells expressing other CYPs had an even stronger effect, with those expressing CYP2B6, CYP5A1, CYP2A13, CYP51A1, or CYP1A2, respectively, being the strongest producers of O2-•.


Assuntos
Sistema Enzimático do Citocromo P-450 , Peróxido de Hidrogênio/metabolismo , Microssomos/enzimologia , Schizosaccharomyces , Superóxidos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética
11.
BMC Plant Biol ; 21(1): 50, 2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33468064

RESUMO

BACKGROUND: Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds. RESULTS: During 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds. CONCLUSIONS: In in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.


Assuntos
Caryophyllales/enzimologia , Lipase/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Triglicerídeos/metabolismo , Caryophyllales/metabolismo , Ésteres/química , Ésteres/metabolismo , Germinação , Hidrólise , Lipase/química , Lipídeos/análise , Lipídeos/química , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Microssomos/metabolismo , Orlistate/farmacologia , Proteínas de Plantas/química , Sementes/enzimologia , Especificidade por Substrato , Temperatura , Triglicerídeos/química , Ceras/química , Ceras/metabolismo
12.
Neurochem Int ; 141: 104884, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33091481

RESUMO

Brain cytochrome P450 (CYP) contributes to the local metabolism of endogenous substrates and drugs. The aim of present study was to ascertain whether the cytochrome P450 2D (CYP2D) activity changes with ageing and in cerebral serotonin deficit. Kinetics of 5-methoxytryptamine O-demethylation to serotonin was studied and the CYP2D activity was measured in brain and liver microsomes of Dark Agouti wild type (WT) rats (mature 3.5-month-old and senescent 21-month-old rats) and in tryptophan hydroxylase 2 (TPH2)-deficient senescent rats. The CYP2D activity and protein level decreased in the frontal cortex of senescent WT rats, but increased in senescent TPH2-deficient rats (compared to senescent WT). In contrast, in the hippocampus, hypothalamus and striatum the CYP2D activity/protein level increased with ageing, but did not change in senescent TPH2-deficient animals (compared to senescent WT). The activity and protein level of liver CYP2D was lower in senescent WT rats than in the mature animals and further decreased in senescent TPH2-deficient rats. In conclusion, ageing and TPH2-deficit affect the CYP2D activity and protein level, which may have a positive impact on neurotransmitter synthesis in brain structures involved in cognitive, emotional or motor functions, but a negative effect on drug metabolism in the liver.


Assuntos
Envelhecimento/metabolismo , Química Encefálica/fisiologia , Encéfalo/enzimologia , Família 2 do Citocromo P450/metabolismo , Fígado/enzimologia , Serotonina/deficiência , Animais , Encéfalo/crescimento & desenvolvimento , Cognição/fisiologia , Emoções/fisiologia , Técnicas de Inativação de Genes , Cinética , Fígado/crescimento & desenvolvimento , Masculino , Microssomos/enzimologia , Microssomos Hepáticos/enzimologia , Ratos , Ratos Wistar , Serotonina/metabolismo , Triptofano Hidroxilase/deficiência , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-32738524

RESUMO

Ethoxyresorufin (ER)-O-deethylation (EROD) activity has been widely used to assess cytochrome P450 1A (CYP1A) activity. The kinetics of CYP1A activity have been well characterized in the liver microsomes. However, studies in kidney microsomes are limited due to the much lower EROD activity in this organ. Here, we developed and validated a sensitive UPLC-MS/MS assay for the characterization of the EROD activity in the rat kidney microsomes. In a 50 µL reaction mixture, rat kidney microsomes (0.25 mg/mL) were incubated with ER (0.1-5 µM) and NADPH (1 mM) for 10 min. Acidic solvents, such as trichloroacetic acid or formic acid, used for quenching of the metabolic reactions and precipitation of the proteins, unexpectedly caused a spontaneous formation of resorufin (RES) from ER. Therefore, the metabolic reactions were terminated by adding acetonitrile, containing a deuterated internal standard (IS). Chromatographic separation was achieved on a C18 UPLC column, and the MS/MS ion transitions were 213.9/185.9 for RES and 220.0/192.0 for IS. The assay was validated in the linear range of 0.5 nM to 75 nM of RES and had a lower limit of quantitation of 0.5 nM. The overall recoveries of RES (90%-99%) and IS (85%-103%) were relatively high, with minimal matrix effect. The assay was successfully applied to the estimation of the Michaelis-Menten (MM) kinetics of EROD activity in the rat kidney microsomes (n = 3), which showed a maximum velocity of 2.68 ± 0.17 pmol/min/mg and a MM constant of 1.72 ± 0.24 µM (mean ± SD). It is concluded that our sensitive and specific analytical method, coupled with the optimized microsomal incubation conditions, provides a robust platform for further investigations of the effects of xenobiotics, environmental factors, or pathophysiologic conditions on the kinetics of EROD activity in the kidney microsomes.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Citocromo P-450 CYP1A1/análise , Rim/metabolismo , Microssomos/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Citocromo P-450 CYP1A1/metabolismo , Rim/citologia , Cinética , Limite de Detecção , Modelos Lineares , Masculino , Microssomos/enzimologia , Oxazinas/análise , Oxazinas/metabolismo , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes
14.
Biochem Pharmacol ; 173: 113639, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31536727

RESUMO

The side effects of nonsteroidal anti-inflammatory drugs (NSAIDs) in the cardiovascular system mainly result from its inhibitory effect on cyclooxygenase-2 (COX-2). Since NSAIDs are one of the most commonly used anti-inflammatory drugs in the clinic, it is necessary to identify new anti-inflammatory drugs that are safer than NSAIDs. Nardosinanone N (NAN), a compound isolated from the roots and rhizomes of Nardostachys chinensis, was evaluated for its anti-inflammatory effects using the lipopolysaccharide (LPS)-stimulated RAW264.7 cell line and rat peritoneal macrophage models. First, we found that NAN down regulated the levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS) and prostaglandin E2 (PGE2), but not cyclooxygenase-2 (COX-2). Additionally, NAN reduced the M1 macrophage phenotype and increased the M2 macrophage phenotype. Furthermore, mechanistic studies showed that NAN activated the nuclear factor-erythroid 2 -related factor 2 (Nrf2) signaling pathway, which, in turn, increased the expression of antioxidant protein heme oxygenase-1 (HO-1) to achieve its anti-inflammatory effect. Finally, Nrf2 siRNA and the HO-1 inhibitor significantly attenuated the anti-inflammatory effect of NAN. More interestingly, we found that NAN did not affect COX-2 expression and activity but reduced the PGE2 concentration by selective inhibition of microsomal prostaglandin E synthase-1 (mPGES-1). In conclusion, NAN may be a new anti-inflammatory drug that has fewer side effects than NSAIDs and can be a new potential Nrf2 activator and mPGES-1 inhibitor.


Assuntos
Compostos de Epóxi/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Nardostachys/química , Preparações de Plantas/farmacologia , Prostaglandina-E Sintases/metabolismo , Terpenos/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Compostos de Epóxi/química , Expressão Gênica/efeitos dos fármacos , Macrófagos/classificação , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Estrutura Molecular , Fator 2 Relacionado a NF-E2/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Preparações de Plantas/química , Prostaglandina-E Sintases/genética , Células RAW 264.7 , Ratos , Transdução de Sinais/efeitos dos fármacos , Terpenos/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
15.
Drug Metab Dispos ; 48(1): 8-17, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31685482

RESUMO

The liver and small intestine restrict oral bioavailability of drugs and constitute the main sites of pharmacokinetic drug-drug interactions. Hence, detailed data on hepatic and intestinal activities of drug metabolizing enzymes is important for modeling drug disposition and optimizing pharmacotherapy in different patient populations. The aim of this study was to determine the activities of seven cytochrome P450 (P450) enzymes in paired liver and small intestinal samples from patients with obesity. Biopsies were obtained from 20 patients who underwent Roux-en-Y gastric bypass surgery following a 3-week low-energy diet. Individual hepatic and intestinal microsomes were prepared and specific probe substrates in combined incubations were used for determination of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A activities. The activities of CYP2C8, CYP2C9, CYP2D6, and CYP3A were quantified in both human liver microsomes (HLM) and human intestinal microsomes (HIM), while the activities of CYP1A2, CYP2B6, and CYP2C19 were only quantifiable in HLM. Considerable interindividual variability was present in both HLM (9- to 23-fold) and HIM (5- to 55-fold). The median metabolic HLM/HIM ratios varied from 1.5 for CYP3A to 252 for CYP2C8. The activities of CYP2C9 in paired HLM and HIM were positively correlated (r = 0.74, P < 0.001), while no interorgan correlations were found for activities of CYP2C8, CYP2D6, and CYP3A (P > 0.05). Small intestinal CYP3A activities were higher in females compared with males (P < 0.05). Hepatic CYP2B6 activity correlated negatively with body mass index (r = -0.72, P < 0.001). These data may be useful for further in vitro-in vivo predictions of drug disposition in patients with obesity. SIGNIFICANCE STATEMENT: Hepatic and intestinal drug metabolism is the key determinant of oral drug bioavailability. In this study, paired liver and jejunum samples were obtained from 20 patients with obesity undergoing gastric bypass surgery following a 3-week low-energy diet. We determined the hepatic and small intestinal activities of clinically important P450 enzymes and provide detailed enzyme kinetic data relevant for predicting in vivo disposition of P450 substrates in this patient population.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Jejuno/enzimologia , Fígado/enzimologia , Microssomos/enzimologia , Obesidade/enzimologia , Índice de Massa Corporal , Sistema Enzimático do Citocromo P-450/genética , Ativação Enzimática , Feminino , Genótipo , Humanos , Técnicas In Vitro , Cinética , Masculino , Microssomos Hepáticos/enzimologia , Sondas Moleculares/metabolismo , Sondas Moleculares/farmacologia , Especificidade de Órgãos , Caracteres Sexuais , Especificidade por Substrato
16.
Artigo em Inglês | MEDLINE | ID: mdl-31669773

RESUMO

The pond wolf spider, Pardosa pseudoannulata, is one of the dominant natural enemies in farmlands and plays important roles in controlling a range of insect pests. The spider is less sensitive to many insecticides than the target pests such as the brown planthopper, Nilaparvata lugens. The different sensitivity to a certain insecticide between species is mostly attributed to the differences in both molecular targets and detoxification enzymes. As one of the most important detoxification enzymes, glutathione transferases (GSTs) play a key role as phase II enzyme in the enzymic detoxification in organisms. Until now, there are few studies on spiders' GSTs, limiting the understanding of insecticide selectivity between insect pests and natural enemy spiders. In this study, based on the transcriptome and genome sequencing of P. pseudoannulata, thirteen full-length transcripts encoding GSTs were identified and analyzed. Interestingly, Delta family, which is thought to be specific to the Insecta, was identified in P. pseudoannulata. Further, vertebrate/mammalian-specific Mu family was also identified in P. pseudoannulata. The mRNA expression levels of cytosolic GSTs in different tissues were determined, and most GST genes were abundant in the gut and the fat body. To investigate GST candidates involving in insecticide detoxification, the mRNA levels of cytosolic GSTs were tested after spiders' exposure to either imidacloprid or deltamethrin. The results showed that PpGSTD3 and PpGSTT1 responded to at least one of these two insecticides. The present study helped understand the function of GSTs in P. pseudoannulata and enriched the genetic information of natural enemy spiders.


Assuntos
Genoma de Inseto , Glutationa Transferase/genética , Aranhas/enzimologia , Sequência de Aminoácidos , Animais , Citosol/enzimologia , Éxons , Corpo Adiposo/enzimologia , Glutationa Transferase/química , Inseticidas/farmacologia , Íntrons , Microssomos/enzimologia , Mitocôndrias/enzimologia , Homologia de Sequência de Aminoácidos , Aranhas/genética
17.
Pestic Biochem Physiol ; 161: 68-76, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31685199

RESUMO

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro cytochrome P450-(P450) mediated oxidative metabolism of xenobiotics, including insecticides. Whereas the preparation of active microsomal membranes from many pest insect species is straightforward, their isolation from honey bees, Apis mellifera (Hymenoptera: Apidae) remained difficult, if not impossible, due to the presence of a yet unidentified endogenous inhibitory factor released during abdominal gut membrane isolation. Thus hampering in vitro toxicological studies on microsomal oxidative phase 1 metabolism of xenobiotics, including compounds of ecotoxicological concern. The use of microsomal membranes rather than individually expressed P450s offers advantages and allows to develop a better understanding of phase 1 driven metabolic fate of foreign compounds. Here we biochemically investigated the problems associated with the isolation of active honey bee microsomes and developed a method resulting in highly active native microsomal preparations from adult female worker abdomens. This was achieved by removal of the abdominal venom gland sting complex prior to microsomal membrane preparation. Molecular sieve chromatography of the venom sac content leads to the identification of phospholipase A2 as the enzyme responsible for the immediate inhibition of cytochrome P450 activity in microsomal preparations. The substrate specificity of functional honey bee microsomes was investigated with different fluorogenic substrates, and revealed a strong preference for coumarin over resorufin derivatives. Furthermore we were able to demonstrate the metabolism of insecticides by honey bee microsomes using an approach coupled to LC-MS/MS analysis of hydroxylated metabolites. Our work provides access to a new and simple in vitro tool to study honey bee phase 1 metabolism of xenobiotics utilising the entire range of microsomal cytochrome P450s.


Assuntos
Abelhas/metabolismo , Microssomos/metabolismo , Fosfolipases A2/isolamento & purificação , Abdome , Animais , Cumarínicos/metabolismo , Hidroxilação , Microssomos/enzimologia , Neonicotinoides/metabolismo , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Especificidade por Substrato , Xenobióticos/metabolismo
18.
Int J Mol Sci ; 20(20)2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614497

RESUMO

Besides the liver, which has always been considered the major source of endogenous glucose production in all post-absorptive situations, kidneys and intestines can also produce glucose in blood, particularly during fasting and under protein feeding. However, observations gained in different experimental animals have given ambiguous results concerning the presence of the glucose-6-phosphatase system in the small intestine. The aim of this study was to better define the species-related differences of this putative gluconeogenic organ in glucose homeostasis. The components of the glucose-6-phosphatase system (i.e., glucose-6-phosphate transporter and glucose-6-phosphatase itself) were analyzed in homogenates or microsomal fractions prepared from the small intestine mucosae and liver of rats, guinea pigs, and humans. Protein and mRNA levels, as well as glucose-6-phosphatase activities, were detected. The results showed that the glucose-6-phosphatase system is poorly represented in the small intestine of rats; on the other hand, significant expressions of glucose-6-phosphate transporter and of the glucose-6-phosphatase were found in the small intestine of guinea pigs and homo sapiens. The activity of the recently described fructose-6-phosphate transporter-intraluminal hexose isomerase pathway was also present in intestinal microsomes from these two species. The results demonstrate that the gluconeogenic role of the small intestine is highly species-specific and presumably dependent on feeding behavior (e.g., fructose consumption) and the actual state of metabolism.


Assuntos
Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Intestino Delgado/enzimologia , Animais , Frutose/metabolismo , Cobaias , Humanos , Microssomos/enzimologia , Ratos , Especificidade da Espécie
19.
Plant Physiol ; 181(4): 1468-1479, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31619508

RESUMO

In most oilseeds, two evolutionarily unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, are the main contributors to the acylation of diacylglycerols in the synthesis of triacylglycerol. DGAT1 and DGAT2 are both present in the important crop oilseed rape (Brassica napus), with each type having four isoforms. We studied the activities of DGAT isoforms during seed development in microsomal fractions from two oilseed rape cultivars: edible, low-erucic acid (22:1) MONOLIT and nonedible high-erucic acid MAPLUS. Whereas the specific activities of DGATs were similar with most of the tested acyl-CoA substrates in both cultivars, MAPLUS had 6- to 14-fold higher activity with 22:1-CoA than did MONOLIT. Thus, DGAT isoforms with different acyl-CoA specificities are differentially active in the two cultivars. We characterized the acyl-CoA specificities of all DGAT isoforms in oilseed rape in the microsomal fractions of yeast cells heterologously expressing these enzymes. All four DGAT1 isoforms showed similar and broad acyl-CoA specificities. However, DGAT2 isoforms had much narrower acyl-CoA specificities: two DGAT2 isoforms were highly active with 22:1-CoA, while the ability of the other two isoforms to use this substrate was impaired. These findings elucidate the importance, which a DGAT isoform with suitable acyl-CoA specificity may have, when aiming for high content of a particular fatty acid in plant triacylglycerol reservoirs.


Assuntos
Acil Coenzima A/metabolismo , Brassica napus/enzimologia , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Erúcicos/metabolismo , Proteínas de Plantas/metabolismo , Brassica napus/genética , Diacilglicerol O-Aciltransferase/genética , Regulação da Expressão Gênica de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Microssomos/enzimologia , Filogenia , Proteínas de Plantas/genética , Sementes/embriologia , Especificidade por Substrato/genética , Triglicerídeos
20.
Toxicol Lett ; 314: 181-186, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31404594

RESUMO

The cytochrome P450 enzyme (CYP) family includes key enzymes for the metabolism of drugs and xenobiotics. Several animal models have been used to determine the metabolite profile of specific drugs. Among these are porcine microsomes prepared from Göttingen minipigs. However, CYP expression profile in microsomes from this pig breed is unknown. In the present study, we determined the mRNA and protein profiles of a comprehensive selection of CYPs in microsomes prepared from male and female Göttingen minipigs. Using RT-PCR, western blotting and mass spectroscopy, we found that the expression levels of CYP1A, CYP2A and CYP2E1 were significantly higher in females than males. Moreover, some of the transcription factors controlling CYP transcription also showed a sex-dependent expression pattern. Conversely, expression of CYP2B, CYP2D and CYP3A was comparable between sexes. The overall CYP expression distribution showed high similarity with what previously been reported in humans. In conclusion, our results suggest that Göttingen minipigs are a reliable model for studying CYPs.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/enzimologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Isoenzimas , Masculino , Microssomos/enzimologia , Modelos Animais , Fatores Sexuais , Especificidade por Substrato , Suínos , Porco Miniatura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...