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1.
BMC Complement Med Ther ; 20(1): 149, 2020 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-32416725

RESUMO

BACKGROUND: Currently there is a high prevalence of humor disorders such as anxiety and depression throughout the world, especially concerning advanced age patients. Aniba riparia (Nees) Mez. (Lauraceae), popular known as "louro", can be found from the Amazon through Guianas until the Andes. Previous studies have already reported the isolation of alkamide-type alkaloids such as riparin III (O-methyl-N-2,6-dyhydroxy-benzoyl tyramine) which has demonstrated anxiolytic and antidepressant-like effects in high doses by intraperitoneal administration. METHODS: Experimental protocol was conducted in order to analyze the anxiolytic-like effect of riparin III at lower doses by intravenous administration to Wistar rats (Rattus norvegicus) (n = 5). The experimental approach was designed to last 15 days, divided in 3 distinct periods of five days: control, anxiogenic and treatment periods. The anxiolytic-like effect was evaluated by experimental behavior tests such as open field and elevated plus-maze test, combined with urine metabolic footprint analysis. The urine was collected daily and analyzed by 1H NMR. Generated data were statistically treated by Principal Component Analysis in order to detect patterns among the distinct periods evaluated as well as biomarkers responsible for its distinction. RESULTS: It was observed on treatment group that cortisol, biomarker related to physiological stress was reduced, indicating anxiolytic-like effect of riparin III, probably through activation of 5-HT2A receptors, which was corroborated by behavioral tests. CONCLUSION: 1H NMR urine metabolic footprint combined with multivariate data analysis have demonstrated to be an important diagnostic tool to prove the anxiolytic-like effect of riparin III in a more efficient and pragmatic way.


Assuntos
Ansiolíticos/farmacologia , Benzamidas/farmacologia , Hidrocortisona/urina , Lauraceae , Tiramina/análogos & derivados , Administração Intravenosa , Animais , Comportamento Animal , Biomarcadores/urina , Brasil , Aprendizagem em Labirinto , Análise Multivariada , Ratos , Ratos Wistar , Tiramina/farmacologia
2.
PLoS One ; 15(4): e0231976, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324789

RESUMO

The two alkaloids gramine and hordenine have been known for playing a role in the allelopathic ability in barley (Hordeum vulgare L.). These allelochemicals can be both found in leaves and roots in some barley cultivars whereas in others one seems to exclude the other. In this study eighteen accessions of barley from the Middle-East area, one accession from Tibet and the modern spring cultivar Barke, already used as parental donor in a nested associated mapping (NAM) population, were screened for their gramine, hordenine and N-methyltyramine (the direct precursor of hordenine) content in leaves, roots and exudates. Moreover, the toxicity of the three allelochemicals on root growth inhibition on lettuce (Lactuca sativa L.) was evaluated. Results of this study showed the preferential production of gramine and hordenine in leaves and roots, respectively, in the nineteen barley accessions. On the other hand, in the modern barley cultivar Barke, the highest content of hordenine in roots and the general lack of gramine suggests a favored biosynthesis of the former. Gramine was not detected in the root exudates. In additions, different metabolomic profiles were observed in wild relatives compared to modern barley genotypes. The results also showed the phytotoxic effects of the three compounds on root growth of lettuce seedlings, with a reduction in root length and an increase of root surface area and diameter. In conclusion, this study highlighted the impact of the domestication effects on the production and distribution of the two allelopathic alkaloids gramine and hordenine in barley.


Assuntos
Alelopatia , Genótipo , Hordeum/genética , Hordeum/metabolismo , Filogenia , Hordeum/crescimento & desenvolvimento , Alcaloides Indólicos/metabolismo , Metabolômica , Especificidade da Espécie , Tiramina/análogos & derivados , Tiramina/metabolismo
3.
Nucleic Acids Res ; 48(10): 5254-5267, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329781

RESUMO

Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ-heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn't extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.


Assuntos
Biotina/química , Biotinilação , DNA/química , Quadruplex G , Heme/química , RNA/química , Animais , Sequência de Bases , Biotina/análogos & derivados , Drosophila melanogaster , Ligantes , Masculino , Salmão , Espermatozoides , Tiramina/análogos & derivados , Tiramina/química
4.
J Vis Exp ; (156)2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32150172

RESUMO

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling using multiple primary antibodies. To minimize antibody cross-reactivity, multiplex immunostaining using indirect staining requires unlabeled primary antibodies from different host species. However, the appropriate combination of different species antibodies is not always available. Here, we describe a method of using unlabeled primary antibodies from the same host species (e.g., in this case both antibodies are from rabbit) for multiplex immunofluorescence on formalin-fixed paraffin-embedded (FFPE) mouse adrenal sections. This method uses the same procedure and reagents used in the antigen retrieval step to strip the activity of the previously stained primary antibody complex. Slides were stained with the first primary antibody using a general immunostaining protocol followed by a binding step with a biotinylated secondary antibody. Then, an avidin-biotin-peroxidase signal development method was used with fluorophore-tyramide as the substrate. The immunoactivity of the first primary antibody complex was stripped through immersion in a microwaved boiling sodium citrate solution for 8 min. The insoluble fluorophore-tyramide deposition remained on the sample, which allowed the slide to be stained with other primary antibodies. Although this method eliminates most false positive signals, some background from antibody cross-reactivity may remain. If the samples are enriched with endogenous biotin, a peroxidase-conjugated secondary antibody may be used to replace the biotinylated secondary antibody to avoid the false positive from recovered endogenous biotin.


Assuntos
Glândulas Suprarrenais/metabolismo , Anticorpos Monoclonais/imunologia , Corantes Fluorescentes/química , Técnicas Imunoenzimáticas/métodos , Micro-Ondas , Tiramina/análogos & derivados , 3-Hidroxiesteroide Desidrogenases/imunologia , Glândulas Suprarrenais/imunologia , Animais , Biotinilação , Sistema Enzimático do Citocromo P-450/imunologia , Imunofluorescência , Humanos , Camundongos , Peroxidase/metabolismo , Coelhos , Coloração e Rotulagem , Tiramina/metabolismo
5.
Carbohydr Polym ; 236: 116042, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32172856

RESUMO

A platform of enzymatically-crosslinked Collagen/Tyramine hyaluronan derivative (Col/HA-Tyr) hydrogels with tunable compositions and gelation conditions was developed to evaluate the impact of the preparation conditions on their physical, chemical and biological properties. At low HA-Tyr content, hydrogels exhibited a fibrillar structure, with lower mechanical properties compared to pure Col hydrogels. At high HA-Tyr and Horse Radish Peroxydase (HRP) content, a microfibrillar network was formed beside the banded Col fibrils and a synergistic effect of the hybrid structure on mechanical properties was observed. These hydrogels were highly resistant against enzymatic degradation while keeping a high degree of hydration. Unlike HA-Tyr hydrogels, encapsulation of human dermal fibroblasts within Col/HA-Tyr hydrogels allowed for high cell viability. These results showed that high HA-Tyr and HRP concentrations are required to positively impact the physical properties of hydrogels while preserving collagen fibrils. Those Col/HA-Tyr hydrogels appear promising for novel tissue engineering applications following a biomimetic approach.


Assuntos
Materiais Biomiméticos/química , Colágenos Fibrilares/química , Ácido Hialurônico/química , Hidrogéis/química , Animais , Armoracia/enzimologia , Materiais Biomiméticos/síntese química , Sobrevivência Celular/efeitos dos fármacos , Matriz Extracelular/química , Colágenos Fibrilares/síntese química , Colágenos Fibrilares/ultraestrutura , Fibroblastos/efeitos dos fármacos , Peroxidase do Rábano Silvestre/química , Humanos , Ácido Hialurônico/síntese química , Ácido Hialurônico/ultraestrutura , Hidrogéis/síntese química , Peróxido de Hidrogênio/química , Ratos Wistar , Tiramina/análogos & derivados , Tiramina/síntese química
6.
J Agric Food Chem ; 68(7): 1998-2006, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31984737

RESUMO

Hordenine, a natural constituent of germinated barley, is a biased agonist of the dopamine D2 receptor. This pilot study investigated the biokinetics of hordenine and its metabolites in four volunteers consuming beer equal to 0.075 mg hordenine/kg body weight. A new ultrahigh-performance liquid chromatography method coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method determined maximum plasma concentrations of 12.0-17.3 nM free hordenine after 0-60 min. Hordenine phase-II metabolism was first dominated by sulfation, but later by glucuronidation. The elimination half-lives in plasma were 52.7-66.4 min for free hordenine and about 60/80 min longer for hordenine sulfate and hordenine glucuronide. Urinary excretion peaked 2-3.5 h after consumption and accumulated to 3.78 µmol within 24 h, corresponding to 9.9% of the ingested dose. The observed hordenine levels in plasma seem too low to provoke direct interaction with the dopamine D2 receptor related to food reward, but synergistic or additive effects with alcohol or N-methyltyramine may occur.


Assuntos
Cerveja/análise , Agonistas de Dopamina/farmacocinética , Tiramina/análogos & derivados , Adulto , Cromatografia Líquida de Alta Pressão , Agonistas de Dopamina/sangue , Agonistas de Dopamina/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Espectrometria de Massas em Tandem , Tiramina/sangue , Tiramina/farmacocinética , Tiramina/urina , Adulto Jovem
7.
J Chromatogr A ; 1613: 460677, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-31727352

RESUMO

The objective of this paper was to extend comprehensive theoretical and experimental investigations at the molecular level to identify factors responsible for the high selectivity of imprinted sorbents. This knowledge was utilized in a new analytical strategy devoted to the analysis of hordenine in human urine after beer consumption. Among the various polymeric compositions tested, the most effective material was built up from methacrylic acid and ethylene glycol dimethacrylate (MIP1), showing a satisfactory binding capacity (4.44 ±â€¯0.15 µmol g-1) and high specificity towards hordenine (AF = 5.90). The comprehensive analyses of porosity data and surface measurements revealed differences between imprinted polymers. The characterization of binding sites of MIP1 revealed a heterogeneous population with two values of Kd (2.75 and 370 µmol L-1) and two values of Bmax (1.82 and 99 µmol g-1) for higher and lower affinity respectively. The extensive theoretical analyses of interactions between various analytes and the MIP model cavity showed the highest binding energy for hordenine (ΔEB1 = -175.17 kcal mol-1). The method was validated for selectivity, lowest limit of quantification, calibration curve performance, precision, accuracy, matrix effect, carry-over and stability in urine. Extracts were prepared according to guidelines of the European Medicines Agency. The validation criteria were fulfilled, and the method was satisfactorily applied to urine samples collected prior to, and 2 h after, consumption of 2 L of beer, revealing the presence of hordenine at the mean level of 129 ±â€¯27 ng mL-1. Additionally, ability of the sorbent to purify the urine sample was assessed using flow injection analysis tandem mass spectrometry, for comparison with other extraction techniques.


Assuntos
Tiramina/análogos & derivados , Urinálise/métodos , Cerveja , Humanos , Metacrilatos/química , Polímeros/química , Tiramina/análise , Tiramina/isolamento & purificação
8.
Mar Drugs ; 17(9)2019 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-31450549

RESUMO

The cell density-dependent mechanism, quorum sensing (QS), regulates the expression of virulence factors. Its inhibition has been proposed as a promising new strategy to prevent bacterial pathogenicity. In this study, 827 strains from the microbiota of sea anemones and holothurians were screened for their ability to produce quorum-sensing inhibitor (QSI) compounds. The strain M3-10, identified as Vibrio alginolyticus by 16S rRNA gene sequencing, as well as ANIb and dDDH analyses, was selected for its high QSI activity. Bioassay-guided fractionation of the cell pellet extract from a fermentation broth of strain M3-10, followed by LC-MS and NMR analyses, revealed tyramine and N-acetyltyramine as the active compounds. The QS inhibitory activity of these molecules, which was confirmed using pure commercially available standards, was found to significantly inhibit Chromobacterium violaceum ATCC 12472 violacein production and virulence factors, such as pyoverdine production, as well as swarming and twitching motilities, produced by Pseudomonas aeruginosa PAO1. This constitutes the first study to screen QSI-producing strains in the microbiota of anemones and holothurians and provides an insight into the use of naturally produced QSI as a possible strategy to combat bacterial infections.


Assuntos
Antibacterianos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Anêmonas-do-Mar/microbiologia , Tiramina/análogos & derivados , Vibrio alginolyticus/metabolismo , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Biofilmes/efeitos dos fármacos , Chromobacterium/efeitos dos fármacos , Chromobacterium/fisiologia , Indóis/antagonistas & inibidores , Indóis/metabolismo , Microbiota , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Tiramina/isolamento & purificação , Tiramina/farmacologia , Vibrio alginolyticus/química , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/metabolismo
9.
Analyst ; 144(15): 4472-4476, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31257395

RESUMO

RNA G-quadruplexes (rG4s) are important RNA secondary structures considering their significance in regulating numerous cellular processes. Described herein is an rG4 detecting and isolation method, which exploits the complex of rG4 and hemin to mimic peroxidase. In the presence of biotin tyramide and hydrogen peroxide, rG4s can be selectively self-biotinylated and easily isolated from a complex RNA mixture using streptavidin magnetic beads.


Assuntos
Quadruplex G , RNA Catalítico/isolamento & purificação , Materiais Biomiméticos/química , Materiais Biomiméticos/isolamento & purificação , Biotina/análogos & derivados , Biotina/química , Biotinilação , Catálise , Hemina/química , Peróxido de Hidrogênio/química , Fenômenos Magnéticos , Mutação , Oxirredução , Peroxidase/química , RNA Catalítico/química , RNA Catalítico/genética , Estreptavidina/química , Tiramina/análogos & derivados , Tiramina/química
10.
Appl Microbiol Biotechnol ; 103(15): 6271-6285, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161392

RESUMO

Proton magnetic resonance-based metabolomics analysis was performed to determine the global metabolite changes in pathogenic bacterium Pseudomonas aeruginosa PAO1 following exposure to quorum sensing (QS) inhibitor hordenine. Pyocyanin inhibition assay confirmed that hordenine exhibited potent QS inhibitory activity. A total of 40 metabolites were assigned by PMR spectra. Hordenine treatment resulted in the destruction of QS system in P. aeruginosa PAO1 by downregulating the expressions of genes involved in QS. The synthesis of antioxidant enzymes was repressed and the oxidative stress was enhanced due to the dysfunctional QS system of P. aeruginosa PAO1. The enhanced oxidative stress induced by the dysfunctional QS system of P. aeruginosa PAO1 altered the membrane components, enhanced membrane permeability, and disturbed energy metabolism, amino acid metabolism, and nucleotide metabolism, and would ultimately attenuate the pathogenicity of P. aeruginosa PAO1. Hordenine may have promising potential for controlling nosocomial pathogens.


Assuntos
Antibacterianos/metabolismo , Metaboloma , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Tiramina/análogos & derivados , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Metabolômica , Tiramina/metabolismo
11.
Methods Mol Biol ; 2008: 41-55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31124087

RESUMO

Peroxidase-catalyzed proximity labeling is a powerful technique for defining the molecular environment of proteins in vivo. Expressing a protein of interest fused to a modified plant peroxidase (APEX2) allows labeling of nearby polypeptides. Addition of hydrogen peroxide (H2O2) and biotin-tyramide (biotin-phenol) generates short-lived radicals around the peroxidase. Labeling is thus restricted to proteins in close proximity, providing a snapshot of the local environment around the APEX2 fusion protein. Combined with an initial perturbation, progressive changes in interaction partners can be tracked, e.g., after drug treatment. Multiplexed quantitative mass spectrometry permits the parallel analysis of several experimental replicates or of up to 11 time points. Here we describe the denaturing purification of biotin-labeled proteins with magnetic streptavidin beads, and subsequent sample preparation for multiplexed quantitative mass spectrometry. Proximity-labeled proteins are enriched under strong denaturing conditions. Tryptic on-bead digest of purified proteins is combined with tandem mass tag peptide labeling (TMT), alkaline reversed-phase peptide fractionation, and SPS MS3 mass spectrometry. This analysis pipeline enables studies of complex protein environment changes in perturbed biological systems, as well as comparative studies of functional protein proximity in different cell lines. Through multiplexing, hundreds of proteins can be quantified in each experimental condition in parallel.


Assuntos
Ascorbato Peroxidases/química , Proteínas de Plantas/química , Proteômica/métodos , Coloração e Rotulagem/métodos , Animais , Biotina/análogos & derivados , Biotina/química , Biotinilação , Linhagem Celular , Humanos , Peróxido de Hidrogênio/química , Espectrometria de Massas/métodos , Tiramina/análogos & derivados , Tiramina/química
12.
Food Chem Toxicol ; 130: 130-141, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31103739

RESUMO

Laba garlic is a kind of processed garlic products, it is the traditional Chinese food with a long history. In this study, the antitumor, antioxidant and cytotoxic properties of the blue pigment (BP) from Laba garlic were investigated. N-trans-feruloyltyramine (FLA) was isolated and identified from BP. The protective effects of FLA against H2O2-induced oxidative damages in L02 cells were also assessed. The apoptotic effects of FLA were detected by using flow cytometry analysis. Results showed that the tumor growth was significantly suppressed by BP (P<0.05). BP and FLA exhibited remarkable antioxidant activities. L02 cells pretreatment with FLA could significantly fight against the oxidative damage induced by H2O2, inhibit the morphological changes of mitochondria and maintain the integrity of mitochondria. FLA showed proliferation inhibition on HepG2 cells with IC50 value of 194 ±â€¯0.894 µM. After treatment of FLA (320 µM), the results of MTT assay on HepG2 and L02 cells indicated that FLA had selective cytotoxic effects. It suggested a new way of prevention and treatment of tumors and FLA might be a promising candidate in cancer therapy and functional foods.


Assuntos
Antioxidantes/farmacologia , Ácidos Cumáricos/farmacologia , Alho/química , Peróxido de Hidrogênio/toxicidade , Tiramina/análogos & derivados , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Manipulação de Alimentos , Humanos , Masculino , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tiramina/farmacologia
13.
Plant J ; 100(1): 20-37, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31124249

RESUMO

Enzyme promiscuity, a common property of many uridine diphosphate sugar-dependent glycosyltransferases (UGTs) that convert small molecules, significantly hinders the identification of natural substrates and therefore the characterization of the physiological role of enzymes. In this paper we present a simple but effective strategy to identify endogenous substrates of plant UGTs using LC-MS-guided targeted glycoside analysis of transgenic plants. We successfully identified natural substrates of two promiscuous Nicotiana benthamiana UGTs (NbUGT73A24 and NbUGT73A25), orthologues of pathogen-induced tobacco UGT (TOGT) from Nicotiana tabacum, which is involved in the hypersensitive reaction. While in N. tabacum, TOGT glucosylated scopoletin after treatment with salicylate, fungal elicitors and the tobacco mosaic virus, NbUGT73A24 and NbUGT73A25 produced glucosides of phytoalexin N-feruloyl tyramine, which may strengthen cell walls to prevent the intrusion of pathogens, and flavonols after agroinfiltration of the corresponding genes in N. benthamiana. Enzymatic glucosylation of fractions of a physiological aglycone library confirmed the biological substrates of UGTs. In addition, overexpression of both genes in N. benthamiana produced clear lesions on the leaves and led to a significantly reduced content of pathogen-induced plant metabolites such as phenylalanine and tryptophan. Our results revealed some additional biological functions of TOGT enzymes and indicated a multifunctional role of UGTs in plant resistance.


Assuntos
Ácidos Cumáricos/metabolismo , Glucose/metabolismo , Glicosiltransferases/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Sesquiterpenos/metabolismo , Tabaco/genética , Tiramina/análogos & derivados , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Glicosídeos/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Tabaco/metabolismo , Tabaco/virologia , Vírus do Mosaico do Tabaco/fisiologia , Tiramina/metabolismo
14.
Front Immunol ; 10: 577, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30972071

RESUMO

The nuclear hormone receptor retinoic acid receptor-related-orphan-receptor-gamma t (RORγt) is the key transcription factor required for Th17 cell differentiation and for production of IL-17 family cytokines by innate and adaptive immune cells. Dysregulated Th17 immune responses have been associated with the pathogenesis of several inflammatory and autoimmune diseases such as psoriasis, psoriatic arthritis, and ankylosing spondylitis. In this article, we describe the in vitro pharmacology of a potent and selective low molecular weight RORγt inhibitor identified after a structure-based hit-to-lead optimization effort. The compound interfered with co-activator binding to the RORγt ligand binding domain and impaired the transcriptional activity of RORγt as evidenced by blocked IL-17A secretion and RORE-mediated transactivation of a luciferase reporter gene. The inhibitor effectively reduced IL-17A production by human naive and memory T-cells and attenuated transcription of pro-inflammatory Th17 signature genes, such as IL17F, IL22, IL26, IL23R, and CCR6. The compound selectively suppressed the Th17/IL-17 pathway and did not interfere with polarization of other T helper cell lineages. Furthermore, the inhibitor was selective for RORγt and did not modify the transcriptional activity of the closely related family members RORα and RORß. Using human keratinocytes cultured with supernatants from compound treated Th17 cells we showed that pharmacological inhibition of RORγt translated to suppressed IL-17-regulated gene expression in keratinocyte cell cultures. Furthermore, in ex vivo immersion skin cultures our RORγt inhibitor suppressed IL-17A production by Th17-skewed skin resident cells which correlated with reduced human ß defensin 2 expression in the skin. Our data suggests that inhibiting RORγt transcriptional activity by a low molecular weight inhibitor may hold utility for the treatment of Th17/IL-17-mediated skin pathologies.


Assuntos
Interleucina-17/fisiologia , Queratinócitos/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Pele/patologia , Células Th17/fisiologia , Acetatos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/fisiologia , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais/fisiologia , Células Th17/citologia , Tiramina/análogos & derivados , Tiramina/farmacologia
15.
Front Immunol ; 10: 701, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019514

RESUMO

Background: DNA extracellular traps (ETs), released by neutrophils (NETs), or eosinophils (EETs), play a pathogenic role in several autoimmune disorders. However, to date, NETs have never been investigated in bullous pemphigoid (BP) with respect to clinical and immunological activities, both at baseline and at time of relapse which have been characterized with specific IL-17 and IL-23 patterns. Objective: We sought to assess whether ETs were associated with BP as well as the relative contribution of IL-17 axis cytokines to NET induction. Methods: Skin biopsy specimens were obtained from 11 patients with BP. Immuno-detection of neutrophils and eosinophils combined to DNA staining allowed us to investigate the in-situ presence of NETs and EETs using confocal scanning microscopy. NETs release was evaluated ex vivo by stimulating polymorphonuclear cells from BP patients with BP biological fluids in presence of IL-17A and IL-23 or of glucocorticoids. Results: At baseline, ETs were observed in BP lesions at the site of dermal-epidermal cleavage. Despite an important infiltrate of eosinophils, ETs were essentially associated with neutrophils in situ and were not related to BP clinical activity at diagnosis. In situ observation of NETs was associated in 6 among 8 patients with serum capacity of NET induction. Notably both blister fluid and sera from BP patients at diagnosis and at time of relapse could induce NET formation ex vivo. In contrast, a longitudinal investigation showed a decrease of NET formation with time of treatment in patients undergoing remission. Mimicking relapse, complementation of sera from BP patients with ongoing remission with either IL-17A or IL-23 increased NET formation. Conversely, IL-17A inhibited NET formation induced by serum from BP patients with relapse supplemented or not with IL-23. Finally, glucocorticoids also inhibited NET formation ex vivo in BP. Conclusion: NET formation is an associated phenomenon with BP. Furthermore, we showed that IL-23 favored NET formation, whereas the effects of IL-17A are environment dependent. Indeed, IL-17A displayed a protective effect on NET formation when associated with IL-23, showing for the first-time differential effects of these two cytokines in BP.


Assuntos
Armadilhas Extracelulares/imunologia , Interleucina-17/sangue , Subunidade p19 da Interleucina-23/sangue , Penfigoide Bolhoso/imunologia , Acetatos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios/farmacologia , Eosinófilos/imunologia , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Masculino , Metilprednisolona/farmacologia , Pessoa de Meia-Idade , Neutrófilos/imunologia , Penfigoide Bolhoso/sangue , Penfigoide Bolhoso/tratamento farmacológico , Estudos Prospectivos , Recidiva , Pesquisa Médica Translacional , Tiramina/análogos & derivados , Tiramina/farmacologia
16.
Biomed Pharmacother ; 111: 1393-1398, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30841454

RESUMO

BACKGROUND: Citri Reticulatae Pericarpium (CRP), Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF) are all important Citrus species used in traditional Chinese medicines (TCMs) for the treatment of gastrointestinal disorders. Although they have been used since ancient times and are still in use today, the mechanistic basis for their regulation of adrenergic receptors (ARs) is still not clear. PURPOSE: In this study, we aimed to determine the active components and mechanisms of action of CRP, AFI and AF in treating gastrointestinal disorders related to ARs. METHODS: First, the phenethylamine alkaloid components of CRP, AFI and AF were identified and compared across 30 samples of three Citrus species by UPLC-Q/TOF-MS in combination with content difference analysis. Second, the effect of the main active alkaloid component on AR-based gastrointestinal disorders was investigated by an in vivo small intestinal propulsive test and an in vitro relaxing small intestinal smooth muscle activity test. The mechanism of AR regulation of the active alkaloid was further studied by evaluating its effect on relaxing small intestinal smooth muscle in the presence of an inhibitor. Lastly, the enzymes, which played an important role in epinephrine synthesis and AR regulation, were detected by immunohistochemistry. RESULTS: Three phenethylamine AR regulators (N-methyltyramine, synephrine and hordenine) in CRP, AFI and AF were characterized. It was found that N-methyltyramine could relax mouse small intestinal smooth muscle and inhibit small intestinal propulsion. The effect of N-methyltyramine on relaxing small intestinal smooth muscle could be inhibited by a-methyl-l-tyrosine. The enzymes related epinephrine synthesis and AR function were found in the mouse small intestine. The biotransformation process that converts N-methyltyramine to epinephrine was determined. CONCLUSION: The treatment of gastrointestinal disorders of CRP, AFI and AF is associated with their alkaloid component N-methyltyramine via the regulation of ARs, and the mechanism is considered to be the biotransformation of N-methyltyramine to epinephrine by serial synthase, which takes place at the nerves cells in small intestine.


Assuntos
Epinefrina/metabolismo , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/metabolismo , Receptores Adrenérgicos/metabolismo , Tiramina/análogos & derivados , Alcaloides/farmacologia , Animais , Citrus/química , Medicamentos de Ervas Chinesas/farmacologia , Frutas/química , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Masculino , Camundongos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Fenetilaminas/metabolismo , Tiramina/farmacologia
17.
J Physiol Biochem ; 75(3): 263-273, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30919256

RESUMO

Various amino acid (AA) metabolites are used as supplements to facilitate metabolic control and enhance responsiveness of insulin-sensitive tissues. ß-hydroxy-ß-methylbutyrate (HMB) is a leucine metabolite proposed to prevent muscle wasting and to mitigate insulin resistance. Taurine, commonly added to energizing drinks, is a metabolite of methionine and cysteine present in bile juice, and proposed to be involved in lipid digestion and to be pro-lipolytic in adipocytes. N-methyltyramine (NMT) is a phenylalanine metabolite found in orange juices at 0.1-3 ppm while its effects on lipid mobilization remain controversial. Here, the putative lipolytic effects of these AA metabolites were studied and it was tested whether they could enhance insulin antilipolytic response in adipocytes. Release of glycerol and non-esterified fatty acids (NEFAs) was measured after a 2-h incubation of adipocytes obtained from control and diet-induced obese mice or from obese patients. In mouse, none of the tested AA derivatives was lipolytic from 1 µM to 1 mM. These compounds did not improve insulin antilipolytic effect or isoprenaline lipolytic action, except for 1 mM NMT that impaired triacylglycerol breakdown in obese mice. In human adipocytes, HMB and taurine were not lipolytic, while NMT weakly activated glycerol and NEFA release at 1 mM. However, 100 µM NMT impaired isoprenaline-stimulated lipolysis in a manner that was hardly added to insulin antilipolytic effect. Since none of these AA derivatives acutely helped or replaced insulin antilipolytic effect in adipocytes, the present in vitro observations do not support their proposed insulin-sensitizing properties. Moreover, NMT, HMB, and taurine were not notably lipolytic.


Assuntos
Adipócitos , Insulina/metabolismo , Lipólise/efeitos dos fármacos , Taurina/farmacologia , Tiramina/análogos & derivados , Valeratos/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Adulto , Animais , Feminino , Humanos , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Obesidade/metabolismo , Tiramina/farmacologia
18.
Pharmacol Biochem Behav ; 180: 44-51, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30904544

RESUMO

Mental disorders have a multifactorial etiology and stress presents as one of the causal factors. In depression, it is suggested that high cortisol concentration contributes directly to the pathology of this disease. Based on that, the study aims to evaluate the potential antidepressant effect of Riparin IV (Rip IV) in mice submitted to chronic stress model by repeated corticosterone administration. Female Swiss mice were selected into four groups: control (Ctrl), corticosterone (Cort), Riparin IV (Cort + Rip IV) and fluvoxamine (Cort + Flu). Three groups were administrated subcutaneously (SC) with corticosterone (20 mg/kg) during twenty-one days, while the control group received only vehicle. After the fourteenth day, groups were administrated tested drugs: Riparin IV, fluvoxamine or distilled water, by gavage, 1 h after subcutaneous injections. After the final treatment, animals were exposed to behavioral models such as forced swimming test (FST), tail suspension test (TST), open field test (OFT), elevated plus maze (EPM) and sucrose preference test (SPT). The hippocampus was also removed for the determination of BDNF levels. Corticosterone treatment altered all parameters in behavioral tests, leading to a depressive- and anxious-like behavior. Riparin IV and fluvoxamine exhibit antidepressant effect in FST, TST and SPT. In EPM and OFT, treatment displayed anxiolytic effect without alteration of locomotor activity. Corticosterone administration decreased BDNF levels and Riparin IV could reestablish them, indicating that its antidepressant effect may be related to ability to ameliorate hippocampal neurogenesis. These findings suggest that Riparin IV improves the depressive and anxious symptoms after chronic stress and could be a new alternative treatment for patients with depression.


Assuntos
Amidas/farmacologia , Antidepressivos/farmacologia , Ansiedade/induzido quimicamente , Benzamidas/farmacologia , Corticosterona/farmacologia , Depressão/induzido quimicamente , Etilaminas/farmacologia , Tiramina/análogos & derivados , Tiramina/farmacologia , Amidas/administração & dosagem , Amidas/uso terapêutico , Anedonia/fisiologia , Animais , Ansiolíticos/administração & dosagem , Ansiolíticos/farmacologia , Antidepressivos/administração & dosagem , Antidepressivos/uso terapêutico , Ansiedade/tratamento farmacológico , Comportamento Animal/efeitos dos fármacos , Benzamidas/administração & dosagem , Benzamidas/uso terapêutico , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corticosterona/administração & dosagem , Depressão/tratamento farmacológico , Modelos Animais de Doenças , Etilaminas/administração & dosagem , Etilaminas/uso terapêutico , Feminino , Fluvoxamina/administração & dosagem , Fluvoxamina/farmacologia , Preferências Alimentares/fisiologia , Elevação dos Membros Posteriores , Hipocampo/metabolismo , Camundongos , Sacarose , Tiramina/administração & dosagem , Tiramina/uso terapêutico
19.
J Agric Food Chem ; 67(3): 784-795, 2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30609368

RESUMO

Serratia marcescens NJ01 is a pathogenic bacterium isolated from diseased tomato leaves. Here, we report on the development of a tomato- S. marcescens host-pathogen system as a model to evaluate the effects of hordenine on quorum sensing (QS)-mediated pathogenicity under native conditions. Exposure to hordenine at 25, 50, and 100 µg/mL significantly inhibited the production of acyl-homoserine lactones and the formation of biofilms. Hordenine treatment notably enhanced the susceptibility of the preformed biofilms to ciprofloxacin by reducing the production of extracellular polysaccharides, destroying the architecture of biofilms, and changing the permeability of membranes, as evidenced by the scattered appearance and dominant red fluorescence in the combination-treated biofilms. Furthermore, the addition of hordenine affected the production of virulence factors, influenced the intracellular metabolites, and downregulated the expressions of QS- and biofilm-related genes. The plant infection model indicated that hordenine could significantly attenuate the pathogenicity of S. marcescens NJ01 in tomato plants. Thus, hordenine could act as a potential pesticide or pesticide accelerant in treating crop infections.


Assuntos
Antibacterianos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/fisiologia , Tiramina/análogos & derivados , Acil-Butirolactonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Lycopersicon esculentum/microbiologia , Doenças das Plantas/microbiologia , Serratia marcescens/genética , Serratia marcescens/patogenicidade , Tiramina/farmacologia , Virulência/efeitos dos fármacos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
20.
Food Chem ; 276: 745-753, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30409657

RESUMO

The phenethylamine alkaloid hordenine, present in germinated barley, was identified recently as a functionally selective dopamine D2 receptor agonist contributing potentially to the rewarding effects of drinking beer. Here, it was shown that the hordenine precursor N-methyltyramine binds with a similar affinity to the dopamine D2 receptor as hordenine (Ki 31.3 µM) showing also selectivity towards the G protein-mediated pathway over the ß-arrestin pathway. Using a newly developed UHPLC-ESI-MS/MS method to monitor beer production, we demonstrated that hordenine and N-methyltyramine were released continuously from barley malt during mashing and were stable during fermentation and conditioning. The amounts released from different base malt types were in a similar range but tended to be higher from caramel malts. Hordenine and N-methyltyramine concentrations in 24 types of beer varied between 1.05-6.32 and 0.59-4.61 mg/L, respectively. Thus, the human uptake of the alkaloids during beer consumption is in the low milligram range.


Assuntos
Cerveja/análise , Agonistas de Dopamina/análise , Tiramina/análogos & derivados , Animais , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetulus , Agonistas de Dopamina/metabolismo , Fermentação , Análise de Alimentos/métodos , Hordeum/metabolismo , Humanos , Ensaio Radioligante , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Tiramina/análise , Tiramina/metabolismo
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