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2.
Life Sci ; 234: 116751, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415771

RESUMO

AIMS: The present study aims to investigate the impacts of olfactory bulbectomy (OBX) on urinary metabolic profile and tryptophan metabolites in prefrontal cortex (PFC) of rats, and to explore the regulation effects of fluoxetine. MAIN METHODS: OBX model was developed by aspiration of olfactory bulbs. After fluoxetine treatment (10 mg/kg) for 14 days, urine samples were collected and behavior tests were applied. Tryptophan (TRP) metabolites and neurotransmitters in PFC were determined by prominence ultrafast liquid chromatography-QTRAP-mass spectrometry, and tryptophan hydroxylase 2 (TPH2) and indoleamine-2,3-dioxygenase 1 (IDO1) were evaluated by western blot. Urinary metabolites were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry-based metabonomics strategy. KEY FINDING: OBX rats showed hyperlocomotion in open field, hyperactivity in open arm and despair status, and fluoxetine reserved these behavioral abnormalities. The levels of TRP, 5-HIAA, 5-HIAA/5-HT ratio and DA increased, while kynurenine and 5-HT decreased in PFC of OBX rats. The activities of TPH2 and IDO1were inhibited after OBX. Twenty-six altered metabolites were identified as potential biomarkers in OBX rats involved in tryptophan metabolism, gut microbiota metabolism, energy metabolism, purine metabolism, ascorbate and aldarate metabolism, and tyrosine metabolism. Among them, 15 abnormal metabolites were corrected by fluoxetine to some extent. SIGNIFICANCE: Our results revealed that urinary metabolic profile changed greatly in OBX rats, and identified biomarkers might be helpful for the diagnosis of agitated depression. The regulation effects of fluoxetine on urinary metabolic profile and tryptophan metabolites in PFC might contribute to its antidepressant action in OBX rats.


Assuntos
Antidepressivos de Segunda Geração/uso terapêutico , Depressão/tratamento farmacológico , Depressão/metabolismo , Fluoxetina/uso terapêutico , Metaboloma/efeitos dos fármacos , Animais , Antidepressivos de Segunda Geração/farmacologia , Depressão/urina , Modelos Animais de Doenças , Fluoxetina/farmacologia , Masculino , Bulbo Olfatório/cirurgia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos Sprague-Dawley , Triptofano/metabolismo
3.
Genes Dev ; 33(17-18): 1236-1251, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31416966

RESUMO

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.


Assuntos
Neoplasias do Colo/fisiopatologia , Cinurenina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Triptofano/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Antineoplásicos/farmacologia , Arilformamidase/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/farmacologia , Cinurenina/genética , Transportador 1 de Aminoácidos Neutros Grandes/genética , Antígenos de Histocompatibilidade Menor/genética , Oximas/farmacologia , Sulfonamidas/farmacologia
4.
Cancer Sci ; 110(10): 3061-3067, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31444833

RESUMO

Tryptophan metabolism is important to induce immune tolerance in tumors. To date, 3 types of tryptophan-metabolizing enzymes have been identified: indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and IDO2) and tryptophan 2,3-dioxygenase 2. Numerous studies have focused on IDO1 as its expression is enhanced in various cancers. Recently, IDO2 has been identified as a tryptophan-metabolizing enzyme that is involved in several immune functions and expressed in cancers such as pancreatic cancer. However, the biological role of IDO2 in the induction of immune tolerance in tumors has not yet been reported. In the present study, we examined the effects of Ido2 depletion on tumor growth in a mouse model of Lewis lung carcinoma by using Ido2-knockout mice. Ido2-knockout mice had reduced tumor volumes compared to WT mice. Furthermore, Ido2 depletion altered the tumor microenvironment, such as tryptophan accumulation and kynurenine reduction, leading to enhancement of immune cell invasion. Finally, enzyme-linked immunospot assay revealed that Ido2 depletion enhanced γ-interferon secretion in the tumor. In conclusion, Ido2 is an important immune regulator in the tumor microenvironment. Our data indicate that IDO2 is a potential target for cancer treatment and drug development.


Assuntos
Carcinoma Pulmonar de Lewis/terapia , Técnicas de Inativação de Genes/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interferon gama/metabolismo , Animais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Cinurenina/metabolismo , Masculino , Camundongos , Triptofano/metabolismo , Carga Tumoral , Evasão Tumoral , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Anim Sci ; 97(8): 3274-3285, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363781

RESUMO

Tryptophan (Trp) is an indispensable amino acid (AA) for dogs of all life stages; however, although Trp requirements for growing dogs are derived from 3 dose-response studies, there are no empirical data on Trp requirements for adult dogs at maintenance. The study objective was to determine Trp requirements of adult dogs of 3 different breeds using the indicator amino acid oxidation (IAAO) technique. Four spayed or neutered Miniature Dachshunds (5.28 ± 0.29 kg BW), 4 spayed Beagles (9.32 ± 0.41 kg BW), and 5 neutered Labrador Retrievers (30.51 ± 2.09 kg BW) were used. After a 14-d adaptation to a Trp-adequate basal diet (Trp = 0.482% dry matter), all dogs were fed a mildly Trp-deficient diet for 2 d (Trp = 0.092% dry matter) before being randomly allocated to receiving 1 of 7 concentrations of Trp supplementation (final Trp content in experimental diets was 0.092, 0.126, 0.148, 0.182, 0.216, 0.249, and 0.283% dry matter) and all dogs received all Trp treatments. After 2-d adaptation to the experimental diets, dogs underwent individual IAAO studies. Total feed was divided in 13 equal meals; at the sixth meal, dogs were fed a bolus of L-[1-13C]-Phenylalanine (Phe) (9.40 mg/kg BW), and thereafter, L-[1-13C]-Phe was supplied (2.4 mg/kg BW) with every meal. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total production of CO2 measured using indirect calorimetry. The maintenance requirement for Trp and the 95% confidence interval (CI) were determined using a 2-phase linear regression model. Mean Trp requirements were estimated at 0.154, 0.218, and 0.157% (dry-matter) for Dachshunds, Beagles, and Labradors, respectively. The upper 95% CI were 0.187, 0.269, and 0.204% (dry-matter) for Dachshunds, Beagles, and Labradors. In conclusion, estimated Trp requirements are higher for Beagles compared with Labradors or Dachshunds, and all estimated requirements are higher than those currently recommended by the NRC and AAFCO.


Assuntos
Aminoácidos/metabolismo , Cães/fisiologia , Necessidades Nutricionais , Triptofano/metabolismo , Aminoácidos Essenciais/metabolismo , Animais , Tamanho Corporal , Peso Corporal , Calorimetria Indireta/veterinária , Dieta/veterinária , Feminino , Masculino , Oxirredução , Fenilalanina/metabolismo , Distribuição Aleatória , Especificidade da Espécie
6.
Chem Commun (Camb) ; 55(58): 8390-8393, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31257394

RESUMO

Diverse bioactive alkaloids with a tryptophan 2,5-diketopiperazine (DKP) core and an annulated structure forming a methylated pyrroloindoline-DKP assembly have been isolated from various microbial sources. However, little is known about their biosynthesis. In this study, a novel indole C3 methyltransferase from Streptomyces sp. HPH0547 was discovered and characterized. Structural elucidation of the products revealed that this enzyme catalyzed unique pyrroloindoline cyclization in tryptophan-containing cyclodipeptides. This is the first C3 methyltransferase reported to catalyze pyrroloindoline cyclization in cyclic dipeptides, which provides a feasible and simple method to access diverse alkaloids.


Assuntos
Alcaloides/biossíntese , Proteínas de Bactérias/metabolismo , Dipeptídeos/biossíntese , Metiltransferases/metabolismo , Peptídeos Cíclicos/biossíntese , Streptomyces/enzimologia , Ciclização , Dicetopiperazinas/metabolismo , Modelos Químicos , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo
7.
J Agric Food Chem ; 67(29): 8186-8190, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31272146

RESUMO

Indole-3-acetic acid (IAA) is considered the most common and important naturally occurring auxin in plants and a major regulator of plant growth and development. In this study, an aldehyde dehydrogenase AldH from Escherichia coli was found to convert indole-3-acetylaldehyde into IAA. Then we established an artificial pathway in engineered E. coli for microbial production of IAA from glucose. The overall pathway includes the upstream pathway from glucose to L-tryptophan and the downstream pathway from L-tryptophan to IAA. To our knowledge, this is the first report on the biosynthesis of IAA directly from a renewable carbon source. The study described here shows the way for the development of a beneficial microbe for biosynthesis of auxin and promoting plant growth in the future.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Indolacéticos/metabolismo , Vias Biossintéticas , Engenharia Genética , Glucose/metabolismo , Triptofano/metabolismo
8.
Food Chem ; 297: 124924, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253284

RESUMO

Yeast nitrogen metabolism produces metabolites, whose origin in wines has scarcely been studied, with an important biological and organoleptic role. The present work focuses on comparing three intracellular extraction methods in order to elucidate efficiency of extraction while measuring the effect of temperature upon the integrity of the compounds related to the metabolism of tryptophan and tyrosine by yeast. Two UHPLC/HRMS methods to measure 16 metabolites were developed and validated. The validation provided optimum values of LOD (7.4·10-6 to 0.1 µg L-1), of LOQ (2·10-5 to 0.02 µg L-1) of precision (11-0.5% RSD) and repeatability (12-0.5% RSD). The removal of interfering molecules enabled matrix effects to be kept at low levels. The results pointed out that the low-temperature methods were more effective, providing better precision for 16 metabolites. The high-temperature extraction method may yield false enhanced compounds concentrations since they originate in cell wall macromolecules degradation.


Assuntos
Fracionamento Químico/métodos , Saccharomyces cerevisiae/metabolismo , Triptofano/análise , Tirosina/análise , Cromatografia Líquida de Alta Pressão , Temperatura Baixa , Congelamento , Glicerol/química , Espectrometria de Massas , Nitrogênio/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo , Vinho/análise
9.
J Agric Food Chem ; 67(26): 7315-7324, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31184122

RESUMO

A high-fat diet (HFD) is the main cause of metabolic diseases. However, HFD in previous studies consists of much lard, which contains a large amount of omega-6 (ω-6) polyunsaturated fatty acid (PUFA) and little omega-3 (ω-3) PUFA. The role of ω-6/ω-3 ratio of HFD in the development of metabolic diseases remains incompletely discussed. In this study, rats were fed with either a low or a high ω-6/ω-3 ratio HFD singly or combined with inulin. Metabolism state was valued and metabolomics of cecal content were detected. Results show that HFD with low ω-6/ω-3 ratio promotes the glucose utilization in rats. However, inulin had different effects on metabolism with different diets. Xanthosine and kynurenic acid in cecum were positively related to epididymal white adipose tissues (eWAT) mass. The present study indicates the beneficial effects of low ω-6/ω-3 ratio HFD (LRD) on the metabolic state of rats. Moreover, xanthosine and kynurenic acid were closely related to the development of metabolic diseases.


Assuntos
Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Mucosa Intestinal/metabolismo , Inulina/metabolismo , Doenças Metabólicas/dietoterapia , Purinas/metabolismo , Triptofano/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Dieta Hiperlipídica , Ácidos Graxos Ômega-3/análise , Ácidos Graxos Ômega-6/análise , Glucose/metabolismo , Humanos , Masculino , Doenças Metabólicas/metabolismo , Ratos , Ratos Sprague-Dawley
10.
Scand J Trauma Resusc Emerg Med ; 27(1): 56, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118076

RESUMO

BACKGROUND: We wanted to define metabolomic patterns in plasma to predict a negative outcome in severe trauma patients. METHODS: A prospective pilot study was designed to evaluate plasma metabolomic patterns, established by liquid chromatography coupled to mass spectrometry, in patients allocated to an intensive care unit (in the University Hospital Arnau de Vilanova, Lleida, Spain) in the first hours after a severe trauma (n = 48). Univariate and multivariate statistics were employed to establish potential predictors of mortality. RESULTS: Plasma of patients non surviving to trauma (n = 5) exhibited a discriminating metabolomic pattern, involving basically metabolites belonging to fatty acid and catecholamine synthesis as well as tryptophan degradation pathways. Thus, concentration of several metabolites exhibited an area under the receiver operating curve (ROC) higher than 0.84, including 3-indolelactic acid, hydroxyisovaleric acid, phenylethanolamine, cortisol, epinephrine and myristic acid. Multivariate binary regression logistic revealed that patients with higher myristic acid concentrations had a non-survival odds ratio of 2.1 (CI 95% 1.1-3.9). CONCLUSIONS: Specific fatty acids, catecholamine synthesis and tryptophan degradation pathways could be implicated in a negative outcome after trauma. The metabolomic study of severe trauma patients could be helpful for biomarker proposal.


Assuntos
Catecolaminas/metabolismo , Ácidos Graxos/metabolismo , Redes e Vias Metabólicas , Metabolômica , Índices de Gravidade do Trauma , Triptofano/metabolismo , Adulto , Idoso , Biomarcadores/sangue , Cromatografia Líquida , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Plasma , Estudos Prospectivos , Sensibilidade e Especificidade , Espanha , Resultado do Tratamento , Ferimentos e Lesões
11.
Cancer Immunol Immunother ; 68(7): 1107-1120, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31119318

RESUMO

Glioblastoma (GBM) is one of the most aggressive tumors. Numerous studies in the field of immunotherapy have focused their efforts on identifying various pathways linked with tumor-induced immunosuppression. Recent research has demonstrated that metabolic reprogramming in a tumor can contribute towards immune tolerance. To begin to understand the interface between metabolic remodeling and the immune-suppressive state in GBM, we performed a focused, integrative analysis coupling metabolomics with gene-expression profiling in patient-derived GBM (n = 80) and compared them to low-grade astrocytoma (LGA; n = 28). Metabolic intermediates of tryptophan, arginine, prostaglandin, and adenosine emerged as immuno-metabolic nodes in GBM specific to the mesenchymal and classical molecular subtypes of GBM. Integrative analyses emphasized the importance of downstream metabolism of several of these metabolic pathways in GBM. Using CIBERSORT to analyze immune components from the transcriptional profiles of individual tumors, we demonstrated that tryptophan and adenosine metabolism resulted in an accumulation of Tregs and M2 macrophages, respectively, and was recapitulated in mouse models. Furthermore, we extended these findings to preclinical models to determine their potential utility in defining the biologic and/or immunologic consequences of the identified metabolic programs. Collectively, through integrative analysis, we uncovered multifaceted ways by which metabolic reprogramming may contribute towards immune tolerance in GBM, providing the framework for further investigations designed to determine the specific immunologic consequence of these metabolic programs and their therapeutic potential.


Assuntos
Neoplasias Encefálicas/imunologia , Glioblastoma/imunologia , Redes e Vias Metabólicas/imunologia , Metaboloma/imunologia , Adenosina/metabolismo , Adulto , Animais , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Triptofano/metabolismo
12.
Prep Biochem Biotechnol ; 49(8): 790-799, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31140364

RESUMO

The sweet-tasting protein brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize brazzein as a sweetener, optimization of fermentation and purification procedure is necessary. Here, we report the expression conditions of brazzein in the yeast Kluyveromices lactis and purification method for maximum yield. Transformed K. lactis was cultured in YPGlu (pH 7.0) at 25 °C and induced by adding glucose:galactose at a weight ratio of 1:2 (%/%) during the stationary phase, which increased brazzein expression 2.5 fold compared to the previous conditions. Cultures were subjected to heat treatment at 80 °C for 1 h, and brazzein containing supernatant was purified using carboxymethyl-sepharose cation exchange chromatography using 50 mM NaCl in 50 mM sodium acetate buffer (pH 4.0) as a wash buffer and 400 mM NaCl (pH 7.0) for elution. The yield of purified brazzein under these conditions was 2.0-fold higher than that from previous purification methods. We also determined that the NanoOrange assay was a suitable method for quantifying tryptophan-deficient brazzein. Thus, it is possible to obtain pure recombinant brazzein with high yield in K. lactis using our optimized expression, purification, and quantification protocols, which has potential applications in the food industry.


Assuntos
Clonagem Molecular/métodos , Microbiologia Industrial/métodos , Kluyveromyces/genética , Proteínas de Plantas/genética , Edulcorantes/metabolismo , Vetores Genéticos/genética , Humanos , Kluyveromyces/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Edulcorantes/análise , Paladar , Triptofano/análise , Triptofano/genética , Triptofano/metabolismo
13.
Appl Microbiol Biotechnol ; 103(12): 4801-4812, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30993383

RESUMO

Poly(ethylene terephthalate) (PET) is one of the most widely applied synthetic polymers, but its hydrophobicity is challenging for many industrial applications. Biotechnological modification of PET surface can be achieved by PET hydrolyzing cutinases. In order to increase the adsorption towards their unnatural substrate, the enzymes are fused to carbohydrate-binding modules (CBMs) leading to enhanced activity. In this study, we identified novel PET binding CBMs and characterized the CBM-PET interplay. We developed a semi-quantitative method to detect CBMs bound to PET films. Screening of eight CBMs from diverse families for PET binding revealed one CBM that possesses a high affinity towards PET. Molecular dynamics (MD) simulations of the CBM-PET interface revealed tryptophan residues forming an aromatic triad on the peptide surface. Their interaction with phenyl rings of PET is stabilized by additional hydrogen bonds formed between amino acids close to the aromatic triad. Furthermore, the ratio of hydrophobic to polar contacts at the interface was identified as an important feature determining the strength of PET binding of CBMs. The interaction of CBM tryptophan residues with PET was confirmed experimentally by tryptophan quenching measurements after addition of PET nanoparticles to CBM. Our findings are useful for engineering PET hydrolyzing enzymes and may also find applications in functionalization of PET.


Assuntos
Metabolismo dos Carboidratos , Carboidratos/química , Interações Hidrofóbicas e Hidrofílicas , Polietilenotereftalatos/metabolismo , Triptofano/metabolismo , Sítios de Ligação , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Ligação Proteica
14.
J Pharm Biomed Anal ; 170: 220-227, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30933897

RESUMO

Indoleamine-2,3-dioxygénase (IDO1) is an enzyme which converts tryptophan (Trp) into kynurenine (Kyn). Having a critical role in tumor immune escape by decreasing Trp and increasing Kyn levels in the microenvironment, IDO1 was one of the first targets for small molecules drug discovery in the field of immuno-oncology. A potent and selective IDO1 inhibitor such as Epacadostat (EPA) was shown to enhance the antitumor activity by restoring the immune system fitness. As exposure at the site of action and to its specific target are identified as the most important factors for success in drug discovery, the objective of this study was to explore the target exposure and intra-tumor pharmacodynamics effects of EPA drug on the tumor metabolism. To do so, we used both Quantitative Mass Spectrometry Imaging (QMSI) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technologies in order to monitor drug and metabolites distribution and their endogenous quantity in the CT26 mouse tumor model. Target exposure analysis showed that almost 61% of EPA signal (26 µg/g) was concentrated within 38% of the entire tumor surface. Semi quantitative analysis of this region confirmed a positive correlation between IDO1 expression and EPA concentration. In parallel, pharmacodynamics analysis highlighted a response efficacy through Kyn/Trp ratio calculation that was shown decreasing after EPA treatment as noticed in treated CT26 tumors (-82%), plasma (-63%) and blood (-62%) compared to control samples. Finally, 15% and 85% of Kyn signal was found in regions with high and low EPA, respectively. In this study, using QMSI, we went further than only quantifying the metabolites and the drug, by estimating the pharmacological effect efficacy of the drug through a target exposure study handled in different regions of the tumor either expressing IDO1 or Kyn.


Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Oximas/farmacologia , Sulfonamidas/farmacologia , Animais , Cromatografia Líquida/métodos , Modelos Animais de Doenças , Cinurenina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Espectrometria de Massas em Tandem/métodos , Triptofano/metabolismo
15.
Int J Oncol ; 54(6): 2127-2138, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942431

RESUMO

R­spondin2 (Rspo2), one of the four members of the R­spondin family of proteins, has agonistic activity in the Wnt/ß­catenin signaling pathway, and it is associated with normal development, as well as disease, such as cancer. The present study focused on the C­mannosylation of Rspo2, which is a novel and unique type of glycosylation that occurs via a C­C linkage between the tryptophan residue and an α­mannose. Although Rspo2 has two putative C­mannosylation sites at residues Trp150 and Trp153, it had not been reported to date whether these sites are C­mannosylated. Firstly, results from mass spectrometry demonstrated that Rspo2 was C­mannosylated at the Trp150 and Trp153 residues. Notably, while this C­mannosylation of Rspo2 resulted in increased extracellular secretion in human fibrosarcoma HT1080 cells, in other human tumor cell lines it inhibited secretion. However, C­mannosylation had consistent effects on the activation of Wnt/ß­catenin signaling in PANC1 and MDA­MB­231 cells, as well as HT1080 cells. Furthermore, overexpression of wild­type Rspo2 significantly increased the migratory ability of A549 and HT1080 cells, whereas overexpression of a C­mannosylation­defective mutant enhanced migration to a lesser degree. These results suggested that C­mannosylation of Rspo2 may promote cancer progression and that the inhibition of C­mannosylation may serve as a potential novel therapeutic approach for cancer therapy.


Assuntos
Movimento Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/patologia , Via de Sinalização Wnt , Linhagem Celular Tumoral , Humanos , Manose/metabolismo , Manosiltransferases/metabolismo , Triptofano/metabolismo , beta Catenina/metabolismo
17.
Nat Commun ; 10(1): 1476, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931940

RESUMO

Early detection and accurate monitoring of chronic kidney disease (CKD) could improve care and retard progression to end-stage renal disease. Here, using untargeted metabolomics in 2155 participants including patients with stage 1-5 CKD and healthy controls, we identify five metabolites, including 5-methoxytryptophan (5-MTP), whose levels strongly correlate with clinical markers of kidney disease. 5-MTP levels decrease with progression of CKD, and in mouse kidneys after unilateral ureteral obstruction (UUO). Treatment with 5-MTP ameliorates renal interstitial fibrosis, inhibits IκB/NF-κB signaling, and enhances Keap1/Nrf2 signaling in mice with UUO or ischemia/reperfusion injury, as well as in cultured human kidney cells. Overexpression of tryptophan hydroxylase-1 (TPH-1), an enzyme involved in 5-MTP synthesis, reduces renal injury by attenuating renal inflammation and fibrosis, whereas TPH-1 deficiency exacerbates renal injury and fibrosis by activating NF-κB and inhibiting Nrf2 pathways. Together, our results suggest that TPH-1 may serve as a target in the treatment of CKD.


Assuntos
Fibrose/metabolismo , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Insuficiência Renal Crônica/metabolismo , Triptofano Hidroxilase/genética , Triptofano/análogos & derivados , Acetilcarnitina/metabolismo , Animais , Canavanina/análogos & derivados , Canavanina/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Estudos de Casos e Controles , Modelos Animais de Doenças , Progressão da Doença , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Humanos , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Rim/citologia , Rim/efeitos dos fármacos , Rim/patologia , Metabolômica , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Taurina/metabolismo , Triptofano/metabolismo , Triptofano/farmacologia , Obstrução Ureteral
18.
Molecules ; 24(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974826

RESUMO

Bacterial indole-3-acetic acid (IAA), an effector molecule in microbial physiology, plays an important role in plant growth-promotion. Here, we comprehensively analyzed about 7282 prokaryotic genomes representing diverse bacterial phyla, combined with root-associated metagenomic data to unravel the distribution of tryptophan-dependent IAA synthesis pathways and to quantify the IAA synthesis-related genes in the plant root environments. We found that 82.2% of the analyzed bacterial genomes were potentially capable of synthesizing IAA from tryptophan (Trp) or intermediates. Interestingly, several phylogenetically diverse bacteria showed a preferential tendency to utilize different pathways and tryptamine and indole-3-pyruvate pathways are most prevalent in bacteria. About 45.3% of the studied genomes displayed multiple coexisting pathways, constituting complex IAA synthesis systems. Furthermore, root-associated metagenomic analyses revealed that rhizobacteria mainly synthesize IAA via indole-3-acetamide (IAM) and tryptamine (TMP) pathways and might possess stronger IAA synthesis abilities than bacteria colonizing other environments. The obtained results refurbished our understanding of bacterial IAA synthesis pathways and provided a faster and less labor-intensive alternative to physiological screening based on genome collections. The better understanding of IAA synthesis among bacterial communities could maximize the utilization of bacterial IAA to augment the crop growth and physiological function.


Assuntos
Bactérias , Genoma Bacteriano , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Planta , Triptofano , Bactérias/genética , Bactérias/metabolismo , Genômica , Reguladores de Crescimento de Planta/genética , Reguladores de Crescimento de Planta/metabolismo , Triptofano/genética , Triptofano/metabolismo
19.
J Agric Food Chem ; 67(18): 5096-5104, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31008593

RESUMO

Tryptophan (Trp) can produce bioactive compounds for appetite regulation, calcium mobilization, and mammary gland homeostasis via a serotonin pathway. This study evaluated the effects of Trp supplementation on the reproduction performance, milk yield, and composition of lactating sows, growth performance of their piglets, and the secretion function of porcine mammary epithelial cells (PMECs). The infrared emulsion analyzer and ELISA analyses revealed that feeding sows with a 0.12% Trp addition increased ( P < 0.05) sow average daily feed intake, milk yield, milk calcium concentration, average daily gain of piglets, fatty acid synthase (FAS) and lactose synthase (LS), ß-casein secretion, intracellular Ca2+ level, the expression of calcium binding protein CaM, and the activity of CaMKII. In a cellular experiment of PMECs treated with Trp, ELISA and flow cytometry analyses revealed that the pretreatment of a Trp hydroxylase inhibitor reduced ( P < 0.05) FAS and LS synthesis, the intracellular Ca2+ level, and the activity of CaMKII. In conclusion, Trp supplementation at 0.12% increased sows' reproductive performance, milk yield, and calcium concentration and piglets' growth performance. Milk yield increased by Trp was linked to 5-hydroxytryptamine-mediated synthesis of FAS, LS, and ß-casein in PMECs, while the increase in calcium concentration was attributed to increasing CaM expression and CAMKII activity.


Assuntos
Leite/química , Suínos/metabolismo , Triptofano/metabolismo , Ração Animal/análise , Animais , Suplementos Nutricionais/análise , Feminino , Lactação , Masculino , Leite/metabolismo , Reprodução , Suínos/crescimento & desenvolvimento
20.
J Pharm Biomed Anal ; 170: 139-152, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30925271

RESUMO

The aim of the presented work was to develop a highly sensitive, accurate and rapid analytical method for the determination of concentration levels of tryptophan and its metabolites of kynurenine catabolic pathway, as well as neurotransmitters and their metabolites in complex biological matrices (brain tissue and blood plasma). The developed analytical method consists of analytes separation from the biological matrices by protein precipitation (blood plasma) or solvent extraction (brain tissue), derivatization of the analytes and their detection by high-performance liquid chromatography combined with mass spectrometry. Individual steps of the whole process were optimized and the method was validated in the terms of selectivity, linearity (R2≥0.980), precision (RSD ≤ 13.3%), recovery (≥82.0%), limit of detection (1.8 ng/mL of blood plasma, 2.2 pg/mg of brain tissue) and limit of quantification (2.5 ng/mL of blood plasma, 2.8 pg/mg of brain tissue). The method was subsequently verified by an animal study, where the concentration levels of the analytes in biological matrices (blood plasma and brain tissue) of T. gondii - infected rats and control animals were compared. All the data obtained from the animal study were statistically evaluated. Increased concentration levels of kynurenine catabolic pathway metabolites (e.g. kynurenine, 3-hydroxykynurenine, quinolinic acid) were observed in the case of T. gondii - infected rats in contrast to the control group. The opposite effect was determined in the case of serotonin and its metabolite 5-hydroxyindoleacetic acid, where higher concentration levels were found in blood plasma of healthy subjects. Finally, Principal Component Analysis (PCA) was utilized for a score plot formation. PCA score plots have demonstrated the similarities of individuals within each group and the differences among the groups.


Assuntos
Encéfalo/metabolismo , Cinurenina/metabolismo , Neurotransmissores/metabolismo , Plasma/metabolismo , Toxoplasmose/sangue , Toxoplasmose/metabolismo , Animais , Cromatografia Líquida de Alta Pressão/métodos , Ácido Hidroxi-Indolacético/sangue , Ácido Hidroxi-Indolacético/metabolismo , Cinurenina/análogos & derivados , Cinurenina/sangue , Masculino , Ácido Quinolínico/sangue , Ácido Quinolínico/metabolismo , Ratos , Ratos Wistar , Serotonina/metabolismo , Espectrometria de Massas em Tandem/métodos , Triptofano/metabolismo
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