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1.
Cell ; 180(1): 6, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31951520

RESUMO

Erythropoietin (EPO) production in the kidney is regulated by the oxygen-sensing transcription factor HIF-1α, which is degraded under normoxic conditions by HIF-prolyl hydroxylase (HIF-PHD). Inhibition of HIF-PHD by roxadustat leads to increased EPO production, better iron absorption, and amelioration of anemia in chronic kidney disease (CKD).


Assuntos
Anemia/terapia , Glicina/análogos & derivados , Fator 1 Induzível por Hipóxia/metabolismo , Isoquinolinas/uso terapêutico , Anemia/metabolismo , Glicina/uso terapêutico , Humanos , Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Prolil Hidroxilases/efeitos dos fármacos , Prolil Hidroxilases/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo
2.
Mol Cell ; 81(9): 2041-2052.e6, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33823141

RESUMO

Cellular senescence is a state of stable proliferative arrest triggered by damaging signals. Senescent cells persist during aging and promote age-related pathologies via the pro-inflammatory senescence-associated secretory phenotype (SASP), whose regulation depends on environmental factors. In vivo, a major environmental variable is oxygenation, which varies among and within tissues. Here, we demonstrate that senescent cells express lower levels of detrimental pro-inflammatory SASP factors in physiologically hypoxic environments, as measured in culture and in tissues. Mechanistically, exposure of senescent cells to low-oxygen conditions leads to AMPK activation and AMPK-mediated suppression of the mTOR-NF-κB signaling loop. Finally, we demonstrate that treatment with hypoxia-mimetic compounds reduces SASP in cells and tissues and improves strength in chemotherapy-treated and aged mice. Our findings highlight the importance of oxygen as a determinant for pro-inflammatory SASP expression and offer a potential new strategy to reduce detrimental paracrine effects of senescent cells.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proliferação de Células , Senescência Celular , Hipóxia/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Fatores Etários , Animais , Antibióticos Antineoplásicos/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Hidroxibenzoatos/farmacologia , Hipóxia/patologia , Hipóxia/fisiopatologia , Mediadores da Inflamação/metabolismo , Isoquinolinas/farmacologia , Camundongos Endogâmicos C57BL , Força Muscular , NF-kappa B/metabolismo , Comunicação Parácrina , Fenótipo , Transdução de Sinais
3.
EMBO J ; 41(1): e105026, 2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34791698

RESUMO

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.


Assuntos
Proteína C9orf72/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Dipeptídeos/metabolismo , Proteólise , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Códon de Iniciação/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expansão das Repetições de DNA/genética , Modelos Animais de Doenças , Drosophila/efeitos dos fármacos , Demência Frontotemporal/patologia , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Isoquinolinas/farmacologia , Longevidade/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Interferência de RNA , Sulfonamidas/farmacologia
4.
Nature ; 577(7790): 432-436, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31915381

RESUMO

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Isoquinolinas/farmacologia , Fenilalanina/análogos & derivados , Piridinas/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Isoquinolinas/química , Cinética , Modelos Moleculares , Fenilalanina/química , Fenilalanina/farmacologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Piridinas/química , Homologia Estrutural de Proteína
5.
Circ Res ; 132(2): e59-e77, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36583384

RESUMO

BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Serina , Camundongos , Animais , Humanos , Isoproterenol/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Serina/metabolismo , Serina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Isoquinolinas/farmacologia , Sulfonamidas/farmacologia , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo
6.
Exp Cell Res ; 439(1): 114098, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38796136

RESUMO

The involvement of γδT cells, Th17 cells, and CD4+CD25+ regulatory T cells (Tregs) is crucial in the progression of pulmonary fibrosis (PF), particularly in maintaining immune tolerance and homeostasis. However, the dynamics of these cells in relation to PF progression, especially under pharmacological interventions, remains poorly understood. This study aims to unravel the interplay between the dynamic changes of these cells and the effect of pharmacological agents in a mouse model of PF induced by intratracheal instillation of bleomycin. We analyzed changes in lung histology, lung index, hydroxyproline levels, and the proportions of γδT cells, Th17 cells, and Tregs on the 3rd, 14th, and 28th days following treatment with Neferine, Isoliensinine, Pirfenidone, and Prednisolone. Our results demonstrate that these drugs can partially or dynamically reverse weight loss, decrease lung index and hydroxyproline levels, and ameliorate lung histopathological damage. Additionally, they significantly modulated the abnormal changes in γδT, Th17, and Treg cell proportions. Notably, on day 3, the proportion of γδT cells increased in the Neferine and Prednisolone groups but decreased in the Isoliensinine and Pirfenidone groups, while the proportion of Th17 cells decreased across all treated groups. On day 14, the Neferine group showed an increase in all three cell types, whereas the Pirfenidone group exhibited a decrease. In the Isoliensinine group, γδT and Th17 cells increased, and in the Prednisolone group, only Tregs increased. By day 28, an increase in Th17 cell proportion was observed in all treatment groups, with a decrease in γδT cells noted in the Neferine group. These shifts in cell proportions are consistent with the pathogenesis changes induced by these anti-PF drugs, suggesting a correlation between cellular dynamics and pharmacological interventions in PF progression. Our findings imply potential strategies for assessing the efficacy and timing of anti-PF treatments based on these cellular changes.


Assuntos
Bleomicina , Fibrose Pulmonar , Linfócitos T Reguladores , Células Th17 , Animais , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Fibrose Pulmonar/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Camundongos , Piridonas/farmacologia , Masculino , Prednisolona/farmacologia , Progressão da Doença , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Pulmão/patologia , Pulmão/imunologia , Pulmão/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Isoquinolinas/farmacologia , Benzilisoquinolinas/farmacologia
7.
Cell Mol Life Sci ; 81(1): 320, 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-39078527

RESUMO

The hypoxia response pathway enables adaptation to oxygen deprivation. It is mediated by hypoxia-inducible factors (HIF), which promote metabolic reprogramming, erythropoiesis, angiogenesis and tissue remodeling. This led to the successful development of HIF-inducing drugs for treating anemia and some of these molecules are now in clinic. However, elevated levels of HIFs are frequently associated with tumor growth, poor prognosis, and drug resistance in various cancers, including hepatocellular carcinoma (HCC). Consequently, there are concerns regarding the recommendation of HIF-inducing drugs in certain clinical situations. Here, we analyzed the effects of two HIF-inducing drugs, Molidustat and Roxadustat, in the well-characterized HCC cell line Huh7. These drugs increased HIF-1α and HIF-2α protein levels which both participate in inducing hypoxia response genes such as BNIP3, SERPINE1, LDHA or EPO. Combined transcriptomics, proteomics and metabolomics showed that Molidustat increased the expression of glycolytic enzymes, while the mitochondrial network was fragmented and cellular respiration decreased. This metabolic remodeling was associated with a reduced proliferation and a lower demand for pyrimidine supply, but an increased ability of cells to convert pyruvate to lactate. This was accompanied by a higher resistance to the inhibition of mitochondrial respiration by antimycin A, a phenotype confirmed in Roxadustat-treated Huh7 cells and Molidustat-treated hepatoblastoma cells (Huh6 and HepG2). Overall, this study shows that HIF-inducing drugs increase the metabolic resilience of liver cancer cells to metabolic stressors, arguing for careful monitoring of patients treated with HIF-inducing drugs, especially when they are at risk of liver cancer.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Carcinoma Hepatocelular , Proliferação de Células , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Isoquinolinas/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos
8.
Proc Natl Acad Sci U S A ; 119(32): e2116289119, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35917342

RESUMO

Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT2R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT2R. We repurposed EMA401, an AT2R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT2R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT2R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or ß-arrestin recruitment. The architecture and interactions of EMA401 in AT2R differ drastically from complexes of AT2R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2-tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.


Assuntos
Bloqueadores do Receptor Tipo 2 de Angiotensina II , Compostos Benzidrílicos , Neoplasias Encefálicas , Reposicionamento de Medicamentos , Glioblastoma , Isoquinolinas , Receptor Tipo 2 de Angiotensina , Analgésicos/farmacologia , Angiotensina II/química , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/uso terapêutico , Apoptose , Compostos Benzidrílicos/química , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Conformação Proteica em alfa-Hélice , Receptor Tipo 2 de Angiotensina/química , Receptor Tipo 2 de Angiotensina/metabolismo , Carga Tumoral/efeitos dos fármacos
9.
Am J Physiol Cell Physiol ; 326(3): C978-C989, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-38314722

RESUMO

Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.


Assuntos
Disfunção Cognitiva , Isoquinolinas , Privação do Sono , Sulfonamidas , Animais , Privação do Sono/tratamento farmacológico , Peixe-Zebra/metabolismo , Cafeína/farmacologia , Rolipram , Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , Cognição , Disfunção Cognitiva/tratamento farmacológico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo
10.
Biochemistry ; 63(5): 660-670, 2024 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-38385972

RESUMO

Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M-1 with a functional "division of labor" among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10-14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.


Assuntos
Acinetobacter baumannii , Isoquinolinas , Sulfonamidas , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Cádmio , Subunidades Proteicas , Espalhamento a Baixo Ângulo , Zinco/metabolismo , Difração de Raios X , Proteínas Repressoras/metabolismo , Metais , DNA/metabolismo
11.
Diabetologia ; 67(9): 1943-1954, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38814443

RESUMO

AIMS/HYPOTHESIS: Hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) enzymes regulate adaptive cellular responses to low oxygen concentrations. Inhibition of HIF-P4Hs leads to stabilisation of hypoxia-inducible factors (HIFs) and activation of the HIF pathway affecting multiple biological processes to rescue cells from hypoxia. As evidence from animal models suggests that HIF-P4H inhibitors could be used to treat metabolic disorders associated with insulin resistance, we examined whether roxadustat, an HIF-P4H inhibitor approved for the treatment of renal anaemia, would have an effect on glucose metabolism in primary human myotubes. METHODS: Primary skeletal muscle cell cultures, established from biopsies of vastus lateralis muscle from men with normal glucose tolerance (NGT) (n=5) or type 2 diabetes (n=8), were treated with roxadustat. Induction of HIF target gene expression was detected with quantitative real-time PCR. Glucose uptake and glycogen synthesis were investigated with radioactive tracers. Glycolysis and mitochondrial respiration rates were measured with a Seahorse analyser. RESULTS: Exposure to roxadustat stabilised nuclear HIF1α protein expression in human myotubes. Treatment with roxadustat led to induction of HIF target gene mRNAs for GLUT1 (also known as SLC2A1), HK2, MCT4 (also known as SLC16A4) and HIF-P4H-2 (also known as PHD2 or EGLN1) in myotubes from donors with NGT, with a blunted response in myotubes from donors with type 2 diabetes. mRNAs for LDHA, PDK1 and GBE1 were induced to a similar degree in myotubes from donors with NGT or type 2 diabetes. Exposure of myotubes to roxadustat led to a 1.4-fold increase in glycolytic rate in myotubes from men with NGT (p=0.0370) and a 1.7-fold increase in myotubes from donors with type 2 diabetes (p=0.0044), with no difference between the groups (p=0.1391). Exposure to roxadustat led to a reduction in basal mitochondrial respiration in both groups (p<0.01). Basal glucose uptake rates were similar in myotubes from donors with NGT (20.2 ± 2.7 pmol mg-1 min-1) and type 2 diabetes (25.3 ± 4.4 pmol mg-1 min-1, p=0.4205). Treatment with roxadustat enhanced insulin-stimulated glucose uptake in myotubes from donors with NGT (1.4-fold vs insulin-only condition, p=0.0023). The basal rate of glucose incorporation into glycogen was lower in myotubes from donors with NGT (233 ± 12.4 nmol g-1 h-1) than in myotubes from donors with type 2 diabetes (360 ± 40.3 nmol g-1 h-1, p=0.0344). Insulin increased glycogen synthesis by 1.9-fold (p=0.0025) in myotubes from donors with NGT, whereas roxadustat did not affect their basal or insulin-stimulated glycogen synthesis. Insulin increased glycogen synthesis by 1.7-fold (p=0.0031) in myotubes from donors with type 2 diabetes. While basal glycogen synthesis was unaffected by roxadustat, pretreatment with roxadustat enhanced insulin-stimulated glycogen synthesis in myotubes from donors with type 2 diabetes (p=0.0345). CONCLUSIONS/INTERPRETATION: Roxadustat increases glycolysis and inhibits mitochondrial respiration in primary human myotubes regardless of diabetes status. Roxadustat may also improve insulin action on glycogen synthesis in myotubes from donors with type 2 diabetes.


Assuntos
Glucose , Glicina , Isoquinolinas , Fibras Musculares Esqueléticas , Humanos , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Glucose/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Células Cultivadas , Pessoa de Meia-Idade , Glicólise/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Adulto
12.
J Cell Mol Med ; 28(20): e70151, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39422159

RESUMO

The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological in vitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell®-based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our in vitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies.


Assuntos
Células Endoteliais da Veia Umbilical Humana , Troca Materno-Fetal , Placenta , Trofoblastos , Humanos , Feminino , Gravidez , Trofoblastos/metabolismo , Placenta/metabolismo , Transporte Biológico , Células Endoteliais da Veia Umbilical Humana/metabolismo , Técnicas de Cocultura , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Modelos Biológicos , Rodamina 123/metabolismo , Leucina/metabolismo , Inulina/metabolismo , Isoquinolinas
13.
Pflugers Arch ; 476(4): 505-516, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38448727

RESUMO

The sodium/proton exchanger isoform 3 (NHE3) is expressed in the intestine and the kidney, where it contributes to hydrogen secretion and sodium (re)absorption. The roles of this transporter have been studied by the use of the respective knockout mice and by using pharmacological inhibitors. Whole-body NHE3 knockout mice suffer from a high mortality rate (with only ∼30% of mice surviving into adulthood), and based on the expression of NHE3 in both intestine and kidney, some conclusions that were originally derived were based on this rather complex phenotype. In the last decade, more refined models have been developed that added temporal and spatial control of NHE3 expression. For example, novel mouse models have been developed with a knockout of NHE3 in intestinal epithelial cells, tubule/collecting duct of the kidney, proximal tubule of the kidney, and thick ascending limb of the kidney. These refined models have significantly contributed to our understanding of the role of NHE3 in a tissue/cell type-specific manner. In addition, tenapanor was developed, which is a non-absorbable, intestine-specific NHE3 inhibitor. In rat and human studies, tenapanor lowered intestinal Pi uptake and was effective in lowering plasma Pi levels in patients on hemodialysis. Of note, diarrhea is seen as a side effect of tenapanor (with its indication for the treatment of constipation) and in intestine-specific NHE3 knockout mice; however, effects on plasma Pi were not supported by this mouse model which showed enhanced and not reduced intestinal Pi uptake. Further studies indicated that the gut microbiome in mice lacking intestinal NHE3 resembles an intestinal environment favoring the competitive advantage of inflammophilic over anti-inflammatory species, something similar seen in patients with inflammatory bowel disease. This review will highlight recent developments and summarize newly gained insight from these refined models.


Assuntos
Isoquinolinas , Trocadores de Sódio-Hidrogênio , Sódio , Sulfonamidas , Animais , Humanos , Camundongos , Ratos , Camundongos Knockout , Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo
14.
Immunology ; 172(4): 614-626, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38685744

RESUMO

Ionising radiation exposure can lead to acute haematopoietic radiation syndrome. Despite significant advancements in the field of radioprotection, no drugs with high efficacy and low toxicity have yet been approved by the Food and Drug Administration. FG-4592, as a proline hydroxylase inhibitor, may play an important role in radioprotection of the haematopoietic system. Mice were peritoneal injected with FG-4592 or normal saline. After irradiation, the survival time, body weight, peripheral blood cell and bone marrow cell (BMC) count, cell apoptosis, pathology were analysed and RNA-sequence technique (RNA-Seq) was conducted to explore the mechanism of FG-4592 in the haematopoietic system. Our results indicated that FG-4592 improved the survival rate and weight of irradiated mice and protected the spleen, thymus and bone marrow from IR-induced injury. The number of BMCs was increased and protected against IR-induced apoptosis. FG-4592 also promoted the recovery of the blood system and erythroid differentiation. The results of RNA-Seq and Western blot showed that the NF-κB signalling pathway and hypoxia-inducible factor-1 (HIF-1) signalling pathway were upregulated by FG-4592. Meanwhile, RT-PCR results showed that FG-4592 could promote inflammatory response significantly. FG-4592 exhibited radioprotective effects in the haematopoietic system by promoting inflammatory response and targeting the NF-κB, HIF signalling pathway.


Assuntos
Apoptose , Radiação Ionizante , Protetores contra Radiação , Animais , Camundongos , Protetores contra Radiação/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , NF-kappa B/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Sistema Hematopoético/efeitos dos fármacos , Sistema Hematopoético/efeitos da radiação , Síndrome Aguda da Radiação/prevenção & controle , Síndrome Aguda da Radiação/tratamento farmacológico , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Lesões Experimentais por Radiação/prevenção & controle , Lesões Experimentais por Radiação/metabolismo , Irradiação Corporal Total , Glicina/análogos & derivados , Isoquinolinas
15.
Antimicrob Agents Chemother ; 68(5): e0161223, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38602413

RESUMO

Mycetoma is a devastating neglected tropical infection of the subcutaneous tissues. It is caused by fungal and bacterial pathogens recognized as eumycetoma and actinomycetoma, respectively. Mycetoma treatment involves diagnosing the causative microorganism as a prerequisite to prescribing a proper medication. Current therapy of fungal eumycetoma causative agents, such as Madurella mycetomatis, consists of long-term antifungal medication with itraconazole followed by surgery, yet with usually unsatisfactory clinical outcomes. Actinomycetoma, on the contrary, usually responds to treatment with co-trimoxazole and amikacin. Therefore, there is a pressing need to discover novel broad-spectrum antimicrobial agents to circumvent the time-consuming and costly diagnosis. Using the resazurin assay, a series of 23 naphthylisoquinoline (NIQ) alkaloids and related naphthoquinones were subjected to in vitro screening against two fungal strains of M. mycetomatis and three bacterial strains of Actinomadura madurae and A. syzygii. Seven NIQs, mostly dimers, showed promising in vitro activities against at least one strain of the mycetoma-causative pathogens, while the naphthoquinones did not show any activity. A synthetic NIQ dimer, 8,8'''-O,O-dimethylmichellamine A (18), inhibited all tested fungal and bacterial strains (IC50 = 2.81-12.07 µg/mL). One of the dimeric NIQs, michellamine B (14), inhibited a strain of M. mycetomatis and significantly enhanced the survival rate of Galleria mellonella larvae infected with M. mycetomatis at concentrations of 1 and 4 µg/mL, without being toxic to the uninfected larvae. As a result, broad-spectrum dimeric NIQs like 14 and 18 with antimicrobial activity are considered hit compounds that could be worth further optimization to develop novel lead antimycetomal agents.


Assuntos
Alcaloides , Antifúngicos , Madurella , Testes de Sensibilidade Microbiana , Micetoma , Micetoma/tratamento farmacológico , Micetoma/microbiologia , Antifúngicos/farmacologia , Animais , Alcaloides/farmacologia , Alcaloides/química , Madurella/efeitos dos fármacos , Isoquinolinas/farmacologia , Actinomadura/efeitos dos fármacos , Naftoquinonas/farmacologia , Larva/microbiologia , Larva/efeitos dos fármacos , Mariposas/microbiologia
16.
Am J Physiol Gastrointest Liver Physiol ; 326(5): G543-G554, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38252683

RESUMO

The pathogenesis of irritable bowel syndrome (IBS) is multifactorial, characterized in part by increased intestinal permeability, and visceral hypersensitivity. Increased permeability is associated with IBS severity and abdominal pain. Tenapanor is FDA-approved for the treatment of IBS with constipation (IBS-C) and has demonstrated improvements in bowel motility and a reduction in IBS-related pain; however, the mechanism by which tenapanor mediates these functions remains unclear. Here, the effects of tenapanor on colonic pain signaling and intestinal permeability were assessed through behavioral, electrophysiological, and cell culture experiments. Intestinal motility studies in rats and humans demonstrated that tenapanor increased luminal sodium and water retention and gastrointestinal transit versus placebo. A significantly reduced visceral motor reflex (VMR) to colonic distension was observed with tenapanor treatment versus vehicle in two rat models of visceral hypersensitivity (neonatal acetic acid sensitization and partial restraint stress; both P < 0.05), returning VMR responses to that of nonsensitized controls. Whole cell voltage patch-clamp recordings of retrogradely labeled colonic dorsal root ganglia (DRG) neurons from sensitized rats found that tenapanor significantly reduced DRG neuron hyperexcitability to capsaicin versus vehicle (P < 0.05), an effect not mediated by epithelial cell secretions. Tenapanor also attenuated increases in intestinal permeability in human colon monolayer cultures caused by incubation with proinflammatory cytokines (P < 0.001) or fecal supernatants from patients with IBS-C (P < 0.005). These results support a model in which tenapanor reduces IBS-related pain by strengthening the intestinal barrier, thereby decreasing permeability to macromolecules and antigens and reducing DRG-mediated pain signaling.NEW & NOTEWORTHY A series of nonclinical experiments support the theory that tenapanor inhibits IBS-C-related pain by strengthening the intestinal barrier. Tenapanor treatment reduced visceral motor responses to nonsensitized levels in two rat models of hypersensitivity and reduced responses to capsaicin in sensitized colonic nociceptive dorsal root ganglia neurons. Intestinal permeability experiments in human colon monolayer cultures found that tenapanor attenuates increases in permeability induced by either inflammatory cytokines or fecal supernatants from patients with IBS-C.


Assuntos
Síndrome do Intestino Irritável , Isoquinolinas , Sulfonamidas , Humanos , Ratos , Animais , Síndrome do Intestino Irritável/tratamento farmacológico , Colo/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Função da Barreira Intestinal , Capsaicina/farmacologia , Células Receptoras Sensoriais/metabolismo , Dor Abdominal/metabolismo , Citocinas/metabolismo , Canais de Cátion TRPV/metabolismo
17.
Oncologist ; 29(3): 272-274, 2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38243388

RESUMO

Duvelisib, a small-molecule phosphatidylinositol 3-kinase-δ,γ inhibitor, has shown efficacy for mycosis fungoides (MF) at dosage ranges of 25-100 mg twice daily (BID), but with significant toxicity. We conducted a retrospective cohort study of patients with advanced MF treated with low-dose duvelisib (15 mg every other day to BID), in an effort to minimize toxicity. A total of 7 patients were included. The overall response rate on duvelisib was 71%, with the remaining patients maintaining stable disease. Mean modified Severity Weighted Assessment Tool score improved by 57.4% and mean percent body surface area involved improved by 52%. Median progression-free survival was 10.3 months. Adverse events occurred in 4 of 7 patients, the most common being fatigue (2/7; grades 1-2), nausea (2/7; grades 1-2), and transaminitis (2/7; grade 3). Overall, low-dose duvelisib showed efficacy for advanced MF with less toxicity, providing a rationale for its use as monotherapy and potentially combinatorial therapy.


Assuntos
Micose Fungoide , Purinas , Neoplasias Cutâneas , Humanos , Estudos Retrospectivos , Micose Fungoide/tratamento farmacológico , Micose Fungoide/induzido quimicamente , Isoquinolinas/efeitos adversos , Neoplasias Cutâneas/tratamento farmacológico
18.
Ann Oncol ; 35(8): 707-717, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38729567

RESUMO

BACKGROUND: SERENA-1 (NCT03616587) is a phase I, multi-part, open-label study of camizestrant in pre- and post-menopausal women with estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer. Parts A and B aim to determine the safety and tolerability of camizestrant monotherapy and define doses for clinical evaluation. PATIENTS AND METHODS: Women aged ≥18 years with metastatic or recurrent ER+, HER2- breast cancer, refractory (or intolerant) to therapy, were assigned 25 mg up to 450 mg once daily (QD; escalation) or 75, 150, or 300 mg QD (expansion). Safety and tolerability, antitumor efficacy, pharmacokinetics, and impact on mutations in the estrogen receptor gene (ESR1m) circulating tumor (ct)DNA levels were assessed. RESULTS: By 9 March 2021, 108 patients received camizestrant monotherapy at 25-450 mg doses. Of these, 93 (86.1%) experienced treatment-related adverse events (TRAEs), 82.4% of which were grade 1 or 2. The most common TRAEs were visual effects (56%), (sinus) bradycardia (44%), fatigue (26%), and nausea (15%). There were no TRAEs grade 3 or higher, or treatment-related serious adverse events at doses ≤150 mg. Median tmax was achieved ∼2-4 h post-dose at all doses investigated, with an estimated half-life of 20-23 h. Efficacy was observed at all doses investigated, including in patients with prior cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) and/or fulvestrant treatment, with and without baseline ESR1 mutations, and with visceral disease, including liver metastases. CONCLUSIONS: Camizestrant is a next-generation oral selective ER antagonist and degrader (SERD) and pure ER antagonist with a tolerable safety profile. The pharmacokinetics profile supports once-daily dosing, with evidence of pharmacodynamic and clinical efficacy in heavily pre-treated patients, regardless of ESR1m. This study established 75-, 150-, and 300-mg QD doses for phase II testing (SERENA-2, NCT04214288 and SERENA-3, NCT04588298).


Assuntos
Neoplasias da Mama , Receptor ErbB-2 , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Idoso , Adulto , Receptores de Estrogênio/metabolismo , Administração Oral , Receptor alfa de Estrogênio/genética , Idoso de 80 Anos ou mais , Dose Máxima Tolerável , Relação Dose-Resposta a Droga , Azetidinas , Isoquinolinas
19.
Cell Physiol Biochem ; 58(4): 445-457, 2024 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-39230349

RESUMO

BACKGROUND/AIMS: Lemons (Citrus limon ) contain various nutrients and are among the most popular citrus fruit. Besides their antioxidant, anticancer, antibacterial, and anti-inflammatory properties, clinical studies have indicated their anti-allergic properties. METHODS: Using the differential-interference contrast (DIC) microscopy, we examined the effects of lemon juice and peel constituents, such as citric acid, ascorbic acid, hesperetin and eriodictyol, on the degranulation from rat peritoneal mast cells. Using fluorescence imaging with a water-soluble dye, Lucifer Yellow, we also examined their effects on the deformation of the plasma membrane. RESULTS: Lemon juice dose-dependently decreased the number of degranulated mast cells. At concentrations equal to or higher than 0.25 mM, citric acid, hesperetin, and eriodictyol significantly reduced the number of degranulating mast cells in a dose-dependent manner, while ascorbic acid required much higher doses to exert significant effects. At 1 mM, citric acid, hesperetin, and eriodictyol almost completely inhibited exocytosis and washed out the Lucifer Yellow trapped on the mast cell surface, while ascorbic acid did not. CONCLUSION: This study provides in vitro evidence for the first time that lemon constituents, such as citric acid, hesperetin, and eriodictyol, potently exert mast cell-stabilizing properties. These properties are attributable to their inhibitory effects on plasma membrane deformation in degranulating mast cells.


Assuntos
Ácido Ascórbico , Citrus , Flavanonas , Hesperidina , Mastócitos , Animais , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Citrus/química , Ratos , Ácido Ascórbico/farmacologia , Masculino , Hesperidina/farmacologia , Hesperidina/química , Flavanonas/farmacologia , Flavanonas/química , Ácido Cítrico/farmacologia , Ácido Cítrico/química , Degranulação Celular/efeitos dos fármacos , Sucos de Frutas e Vegetais/análise , Peritônio/citologia , Ratos Sprague-Dawley , Exocitose/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Frutas/química , Isoquinolinas
20.
BMC Plant Biol ; 24(1): 276, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38605285

RESUMO

BACKGROUND: Stephania kwangsiensis Lo (Menispermaceae) is a well-known Chinese herbal medicine, and its bulbous stems are used medicinally. The storage stem of S. kwangsiensis originated from the hypocotyls. To date, there are no reports on the growth and development of S. kwangsiensis storage stems. RESULTS: The bulbous stem of S. kwangsiensis, the starch diameter was larger at the stable expanding stage (S3T) than at the unexpanded stage (S1T) or the rapidly expanding stage (S2T) at the three different time points. We used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and Illumina sequencing to identify key genes involved in bulbous stem development. A large number of differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) were identified. Based on the differential expression profiles of the metabolites, alkaloids, lipids, and phenolic acids were the top three differentially expressed classes. Compared with S2T, significant changes in plant signal transduction and isoquinoline alkaloid biosynthesis pathways occurred at both the transcriptional and metabolic levels in S1T. In S2T compared with S3T, several metabolites involved in tyrosine metabolism were decreased. Temporal analysis of S1T to S3T indicated the downregulation of phenylpropanoid biosynthesis, including lignin biosynthesis. The annotation of key pathways showed an up-down trend for genes and metabolites involved in isoquinoline alkaloid biosynthesis, whereas phenylpropanoid biosynthesis was not completely consistent. CONCLUSIONS: Downregulation of the phenylpropanoid biosynthesis pathway may be the result of carbon flow into alkaloid synthesis and storage of lipids and starch during the development of S. kwangsiensis bulbous stems. A decrease in the number of metabolites involved in tyrosine metabolism may also lead to a decrease in the upstream substrates of phenylpropane biosynthesis. Downregulation of lignin synthesis during phenylpropanoid biosynthesis may loosen restrictions on bulbous stem expansion. This study provides the first comprehensive analysis of the metabolome and transcriptome profiles of S. kwangsiensis bulbous stems. These data provide guidance for the cultivation, breeding, and harvesting of S. kwangsiensis.


Assuntos
Alcaloides , Plantas Medicinais , Stephania , Stephania/química , Stephania/metabolismo , Plantas Medicinais/metabolismo , Cromatografia Líquida/métodos , Lignina/metabolismo , Espectrometria de Massas em Tandem , Melhoramento Vegetal , Perfilação da Expressão Gênica , Transcriptoma , Alcaloides/metabolismo , Amido/metabolismo , Isoquinolinas/metabolismo , Tirosina/metabolismo , Lipídeos , Regulação da Expressão Gênica de Plantas
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