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1.
Chem Biodivers ; 16(12): e1900365, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31589372

RESUMO

The quest for reliable dihydroorotate dehydrogenase (DHODH) inhibitors has engendered the discovery of potential therapeutic compounds at different stages of clinical trials. Although promising, high attrition rates and unfavorable bioactivities have limited their drug developmental progress. A recent structural modification of DSM265, a triazolopyrimidine-based inhibitor, yielded DSM421, derived by the substitution of the SF5 -aniline group on DSM265 with a CF3 -pyridinyl moiety. Consequently, DSM421 exhibited improved pharmacological and pharmacokinetics attributes relative to DSM265. The improved bioactivity mediated by the CF3 -pyridinyl group leaves us with a curiosity to investigate underlying ligand-binding mechanisms and dynamics using computational methods. Presented in this study are insights that clearly explain the effects of structural SF5 -aniline→CF3 -pyridinyl modifications on pfDHODH inhibition. Findings showed that the CF3 -pyridinyl group induced an optimal and stabilized positioning of DSM421 within the binding pocket, allowing for steady and strong intermolecular interactions which favored its stronger binding affinity as estimated and correlated with bioactivity data. These interactions consequently induced a pronounced stabilization of the structural conformation of pfDHODH by restricting residue motions, which possibly underpinned its enhanced inhibitory activity relative to DSM265. Active site interactions of the CF3 -pyrinidyl group with residues Ser236, Ile237, and Phe188 characterized by strong π-π stacking and halogen interactions also stabilized its positioning which altogether accounted for its enhanced inhibitory prowess towards pfDHODH. On the contrary, fewer and weaker interactions characterized DSM265 binding which could explain its relatively lower binding affinity. Findings will facilitate the design of novel pfDHODH inhibitors with enhanced properties.


Assuntos
Antimaláricos/química , Inibidores Enzimáticos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Purinas/química , Piridinas/química , Antimaláricos/metabolismo , Sítios de Ligação , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Simulação de Dinâmica Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Protozoários/metabolismo , Purinas/metabolismo , Teoria Quântica , Termodinâmica
2.
Int J Mol Sci ; 20(13)2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31284385

RESUMO

Mitochondrial oxidative stress accumulates with aging and age-related diseases and induces alterations in mitochondrial DNA (mtDNA) content. Since mtDNA qualitative alterations are also associated with aging, repair of mtDNA damage is of great importance. The most relevant form of DNA repair in this context is base excision repair (BER), which removes oxidized bases such as 8-oxoguanine (8-oxoG) and thymine glycol through the action of the mitochondrial isoform of the specific 8-oxoG DNA glycosylase/apurinic or apyrimidinic (AP) lyase (OGG1) or the endonuclease III homolog (NTH1). Mouse strains lacking OGG1 (OGG1-/-) or NTH1 (NTH1-/-) were analyzed for mtDNA alterations. Interestingly, both knockout strains presented a significant increase in mtDNA content, suggestive of a compensatory mtDNA replication. The mtDNA "common deletion" was not detected in either knockout mouse strain, likely because of the young age of the mice. Formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites accumulated in mtDNA from OGG1-/- but not from NTH1-/- mice. Interestingly, the D-loop region was most severely affected by the absence of OGG1, suggesting that this region may be a hotspot for oxidative damage. Thus, we speculate that mtDNA alterations may send a stress message to evoke cell changes through a retrograde mitochondrial-nucleus communication.


Assuntos
Dano ao DNA/genética , DNA Glicosilases/genética , DNA Mitocondrial/genética , Deleção de Genes , Purinas/metabolismo , Animais , Pareamento de Bases/genética , Camundongos Knockout , Oxirredução , Deleção de Sequência
4.
J Pharmacol Sci ; 140(1): 109-112, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31155393

RESUMO

Cancer cachexia is a systemic wasting syndrome characterized by anorexia and loss of body weight. The xanthine oxidase (XO) inhibitor febuxostat is one of the promising candidates for cancer cachexia treatment. However, cachexic symptoms were not alleviated by oral administration of febuxostat in our cancer cachexia model. Metabolomic analysis with brains of our cachexic model showed that purine metabolism was activated and XO activity was increased, and thus suggested that febuxostat would not reach the brain. Accordingly, targeting XO in the brain, which controls appetite, may be an effective strategy for treatment of cancer cachexia.


Assuntos
Encéfalo/enzimologia , Encéfalo/metabolismo , Caquexia/tratamento farmacológico , Febuxostat/administração & dosagem , Neoplasias/complicações , Xantina Oxidase/metabolismo , Administração Oral , Animais , Caquexia/enzimologia , Caquexia/etiologia , Caquexia/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos BALB C , Purinas/metabolismo , Xantina Oxidase/fisiologia
5.
Pharmacol Rev ; 71(3): 345-382, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235653

RESUMO

Immune-mediated inflammatory diseases (IMIDs) encompass a wide range of seemingly unrelated conditions, such as multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases, asthma, chronic obstructive pulmonary disease, and systemic lupus erythematosus. Despite differing etiologies, these diseases share common inflammatory pathways, which lead to damage in primary target organs and frequently to a plethora of systemic effects as well. The purinergic signaling complex comprising extracellular nucleotides and nucleosides and their receptors, the P2 and P1 purinergic receptors, respectively, as well as catabolic enzymes and nucleoside transporters is a major regulatory system in the body. The purinergic signaling complex can regulate the development and course of IMIDs. Here we provide a comprehensive review on the role of purinergic signaling in controlling immunity, inflammation, and organ function in IMIDs. In addition, we discuss the possible therapeutic applications of drugs acting on purinergic pathways, which have been entering clinical development, to manage patients suffering from IMIDs.


Assuntos
Inflamação/tratamento farmacológico , Inflamação/imunologia , Agonistas Purinérgicos/farmacologia , Antagonistas Purinérgicos/farmacologia , Purinas/metabolismo , Receptores Purinérgicos/metabolismo , Animais , Humanos , Inflamação/metabolismo , Terapia de Alvo Molecular , Purinas/imunologia , Receptores Purinérgicos/imunologia , Transdução de Sinais/efeitos dos fármacos
6.
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
7.
Bioanalysis ; 11(10): 1003-1013, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31218896

RESUMO

Aim: In order to differential diagnosis of chronic hepatitis B (HBV-I) and hepatocellular carcinoma (HCC), a UPLC-MS/MS method for measuring purine metabolites was developed. Methodology & results: serum samples from 26 HBV-I and 35 HCC patients were collected. Ten purine metabolites were simultaneously quantified by UPLC-MS/MS with tubercidin and uric acid-1,3-15N2 as internal standards. The method was validated to meet the requirements of clinical sample analysis. A logistic equation was established for differential diagnosis of HBV-I and HCC by combination of xanthosine and guanine with the area under the receiver operating characteristic curve of 0.885. Conclusion: Guanine and xanthosine are intermediates in the metabolism of purine, which play an important role in gene synthesis, and metabolism regulation. The alteration of serum purine metabolite may contribute to differential diagnosis of HBV-I and HCC.


Assuntos
Análise Química do Sangue/métodos , Carcinoma Hepatocelular/diagnóstico , Hepatite B Crônica/diagnóstico , Neoplasias Hepáticas/diagnóstico , Purinas/sangue , Purinas/metabolismo , Adulto , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/metabolismo , Cromatografia Líquida de Alta Pressão , Diagnóstico Diferencial , Feminino , Hepatite B Crônica/sangue , Hepatite B Crônica/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/metabolismo , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Curva ROC , Espectrometria de Massas em Tandem
8.
Cells ; 8(6)2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31141888

RESUMO

Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are tandem-type lesions observed among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5'R and 5'S, for each 2'-deoxyadenosine and 2'-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2'-deoxyribose units generating C5' radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the preparation of the cPu molecular library for analytical and DNA synthesis applications for the studies of the enzymatic recognition and repair mechanisms, their impact on transcription and genetic instability, quantitative determination of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols.


Assuntos
Dano ao DNA , Purinas/química , Purinas/metabolismo , Animais , Reparo do DNA , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Espécies Reativas de Oxigênio/metabolismo , Bibliotecas de Moléculas Pequenas
9.
Poult Sci ; 98(10): 4327-4337, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31111951

RESUMO

Consumer preference for slow-growing broiler chickens is rising because of increased demand for high-quality poultry products. Korat chicken (KRC) is a slow-growing chicken generated in Thailand. A goal of the KRC breeding program is to produce meat with a low purine content to benefit an aging population, without interfering with growth performance. Thus, this study aimed to investigate the effects of genes encoding melanocortin 4 receptor (MC4R), calpain 1 (CAPN1), and adenylosuccinate lyase (ADSL) on body weight, muscle fiber, and content of purine and its derivatives (i.e., adenine, guanine, hypoxanthine, and xanthine), to develop molecular markers for breeding programs. Genotypes of MC4R, CAPN1, and ADSL were obtained from 583 KRCs by PCR-single-strand conformation polymorphism. The body weight and purine contents of the KRCs were measured every 2 wk until the KRCs reached market weight at 10 wk of age. A significant association between the MC4R genotype and body weight at 2, 4, and 10 wk of age was detected. KRC possessing the BB genotype of CAPN1 showed significantly heavier body weight at 6 wk of age and guanine content at 4 wk of age, and a smaller muscle fiber diameter in the breast muscle at 10 wk of age, compared with those of the other genotypes. In addition, high expression levels of the CAPN1 and ADSL genes were detected in the breast muscle at 2 wk of age. Although higher purine contents were detected at a young age, no significant associations with the MC4R, CAPN1, and ADSL genes were detected. Our results indicate that MC4R and CAPN1 could be used as genetic markers for growth and meat quality in the slow-growing chicken breeding program.


Assuntos
Proteínas Aviárias/genética , Peso Corporal/genética , Galinhas/fisiologia , Purinas/metabolismo , Adenilossuccinato Liase/genética , Adenilossuccinato Liase/metabolismo , Animais , Proteínas Aviárias/metabolismo , Calpaína/genética , Calpaína/metabolismo , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Feminino , Marcadores Genéticos , Genótipo , Masculino , Carne/análise , Polimorfismo Conformacional de Fita Simples , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo
10.
J Sep Sci ; 42(15): 2523-2533, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31144454

RESUMO

We present an ultra high performance liquid chromatography with ultraviolet spectroscopy and quadrupole time-of-flight mass spectrometry method for the simultaneous quantification of ten purines (adenine, hypoxanthine, guanine, xanthine, deoxyadenosine, adenosine, inosine, guanosine, xanthosine, and uric acid) and creatinine in human urine. After chromatographic separation on an ACE Excel 2 AQ column, high abundant creatinine and uric acid and the other low abundant purines were sequentially detected by ultraviolet and quadrupole time-of-flight mass spectrometry within a single run. Method validations including specificity (improved by accurate mass measurement), linearity (correlation coefficients ≥0.9944), limit of quantification (0.002-9.756 µg/mL), intra- and interday precision (relative standard deviations ≤9.1 and 14.0%, respectively), accuracy (relative errors ≤13.1%), extraction recovery (between 90.3 and 109.6%), matrix effect (between 85.3 and 110.5%), and stability (relative errors ≤14.3%) were fully evaluated. This approach was applied to characterize the disordered purine metabolism in acute and chronic gout as an example. Quantitative results (normalized by creatinine) showed that an overproduction of urinary purine precursors might be involved in the gout process. The developed method represents a useful tool to investigate the purine disturbances in gout and other relevant diseases.


Assuntos
Creatinina/urina , Gota/urina , Purinas/urina , Cromatografia Líquida de Alta Pressão , Creatinina/metabolismo , Humanos , Espectrometria de Massas , Purinas/metabolismo , Espectrofotometria Ultravioleta , Fatores de Tempo
11.
Sci Total Environ ; 676: 72-86, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31029902

RESUMO

BACKGROUND: Environmental pollutants, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), are common surfactants in various consumer products. Epidemiological studies have demonstrated the association of diabetic kidney diseases with PFOA and PFOS. However, mechanisms of metabolic alterations involved are still unclear. METHODS: Considering their involvement of glomerular hemodynamics, rat mesangial cells (MCs) are used as an in vitro model of diabetic kidney diseases for exposure to PFOS/PFOA under diabetic condition. Non-targeted metabolomics studies based on liquid chromatography-high resolution mass spectrometry were conducted to determine how PFOA/PFOS promoted fibrotic and proinflammatory responses in the MCs under diabetic condition. RESULTS: Exposure of PFOA/PFOS (10 µM) increased oxidative stress and the levels of fibrotic and proinflammatory markers in MCs under diabetic condition. We demonstrated for the first time that PFOA and PFOS altered amino acid biosynthesis, citrate cycle, and purine metabolism in MCs under diabetic condition. Compared with diabetic condition, the exposure of PFOA and PFOS under diabetic condition more significantly altered the levels of 13 intracellular metabolites, including L-tyrosine, L-phenylalanine, L-arginine, L-tryptophan, AMP, ADP, UMP, inosine, and hypoxanthine, which have been reported to be related to kidney injury. In addition, PFOA/PFOS treatment significantly altered the expression levels of key enzymes involved in these metabolisms. Treatment with L-tyrosine, L-phenylalanine, L-arginine, and L-tryptophan reduced the levels of fibrotic and inflammatory markers induced by PFOA/PFOS. CONCLUSION: Our results suggest that under diabetic condition, exposure of PFOA or PFOS aggravated diabetic kidney injury in vitro by impairing metabolisms of amino acids and purines to induce more fibrosis and inflammation in MCs.


Assuntos
Ácidos Alcanossulfônicos/toxicidade , Aminoácidos/metabolismo , Caprilatos/toxicidade , Fluorcarbonetos/toxicidade , Purinas/metabolismo , Animais , Diabetes Mellitus/metabolismo , Poluentes Ambientais , Rim/metabolismo , Ratos , Testes de Toxicidade
12.
Can J Microbiol ; 65(8): 596-612, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31018106

RESUMO

Ureaplasma diversum is a member of the Mollicutes class responsible for urogenital tract infection in cattle and small ruminants. Studies indicate that the process of horizontal gene transfer, the exchange of genetic material among different species, has a crucial role in mollicute evolution, affecting the group's characteristic genomic reduction process and simplification of metabolic pathways. Using bioinformatics tools and the STRING database of known and predicted protein interactions, we constructed the protein-protein interaction network of U. diversum and compared it with the networks of other members of the Mollicutes class. We also investigated horizontal gene transfer events in subnetworks of interest involved in purine and pyrimidine metabolism and urease function, chosen because of their intrinsic importance for host colonization and virulence. We identified horizontal gene transfer events among Mollicutes and from Ureaplasma to Staphylococcus aureus and Corynebacterium, bacterial groups that colonize the urogenital niche. The overall tendency of genome reduction and simplification in the Mollicutes is echoed in their protein interaction networks, which tend to be more generalized and less selective. Our data suggest that the process was permitted (or enabled) by an increase in host dependence and the available gene repertoire in the urogenital tract shared via horizontal gene transfer.


Assuntos
Proteínas de Bactérias/metabolismo , Transferência Genética Horizontal , Genoma Bacteriano , Mapas de Interação de Proteínas , Tenericutes/genética , Ureaplasma/genética , Animais , Proteínas de Bactérias/genética , Bovinos , Corynebacterium/genética , Evolução Molecular , Tamanho do Genoma , Genômica , Redes e Vias Metabólicas , Purinas/metabolismo , Pirimidinas/metabolismo , Staphylococcus aureus/genética , Tenericutes/classificação , Tenericutes/metabolismo , Ureaplasma/classificação , Ureaplasma/metabolismo , Virulência
13.
Int J Mol Sci ; 20(9)2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027387

RESUMO

Plant regeneration via somatic embryogenesis (SE) is a key step during genetic engineering. In the current study, integrated widely targeted metabolomics and RNA sequencing were performed to investigate the dynamic metabolic and transcriptional profiling of cotton SE. Our data revealed that a total of 581 metabolites were present in nonembryogenic staged calli (NEC), primary embryogenic calli (PEC), and initiation staged globular embryos (GE). Of the differentially accumulated metabolites (DAMs), nucleotides, and lipids were specifically accumulated during embryogenic differentiation, whereas flavones and hydroxycinnamoyl derivatives were accumulated during somatic embryo development. Additionally, metabolites related to purine metabolism were significantly enriched in PEC vs. NEC, whereas in GE vs. PEC, DAMs were remarkably associated with flavonoid biosynthesis. An association analysis of the metabolome and transcriptome data indicated that purine metabolism and flavonoid biosynthesis were co-mapped based on the Kyoto encyclopedia of genes and genomes (KEGG) database. Moreover, purine metabolism-related genes associated with signal recognition, transcription, stress, and lipid binding were significantly upregulated. Moreover, several classic somatic embryogenesis (SE) genes were highly correlated with their corresponding metabolites that were involved in purine metabolism and flavonoid biosynthesis. The current study identified a series of potential metabolites and corresponding genes responsible for SE transdifferentiation, which provides a valuable foundation for a deeper understanding of the regulatory mechanisms underlying cell totipotency at the molecular and biochemical levels.


Assuntos
Flavonoides/metabolismo , Gossypium/metabolismo , Purinas/metabolismo , Transdiferenciação Celular , Regulação da Expressão Gênica de Plantas/genética , Gossypium/genética , Metaboloma/genética , Metaboloma/fisiologia , Transcriptoma/genética
14.
Curr Genet ; 65(4): 893-897, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30937517

RESUMO

This mini-review considers the idea that guanylate nucleotide energy charge acts as an integrative signal for the regulation of gene expression in eukaryotic cells and discusses possible routes for that signal's transduction. Gene expression is intimately linked with cell nutrition and diverse signaling systems serve to coordinate the synthesis of proteins required for growth and proliferation with the prevailing cellular nutritional status. Using short pathways for the inducible and futile consumption of ATP or GTP in engineered cells of Saccharomyces cerevisiae, we have recently shown that GTP levels can also play a role in determining how genes act to respond to changes in cellular energy supply. This review aims to interpret the importance of GTP as an integrative signal in the context of an increasing body of evidence indicating the spatio-temporal complexity of cellular de novo purine nucleotide biosynthesis.


Assuntos
Metabolismo Energético/genética , Nucleotídeos de Guanina/genética , Transcrição Genética , Purinas/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
15.
Cell Prolif ; 52(3): e12595, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30953394

RESUMO

OBJECTIVES: Mesenchymal stem cells (MSCs) could regulate the function of various immune cells. It remains unclear whether MSCs additionally possess immunostimulatory properties. We investigated the impact of human MSCs on the responsiveness of primary natural killer (NK) cells in terms of induction of anti-inflammatory purinergic signalling. MATERIAL AND METHODS: We obtained human bone marrow mesenchymal stem cells (BMMSCs) and dental pulp stem cells (DPSCs). NK cells were isolated from peripheral blood of healthy volunteers. Activated NK cells were cultured with MSCs. Proliferation assay, apoptosis analysis, activating or inhibitory receptor expression and degranulation assay were used to explore NK cells' function. High-performance liquid chromatography was used to investigate the purinergic signalling in activated NK cells. RESULTS: Both DPSCs and BMMSCs could impair proliferation and promote apoptosis of activated NK cells. Also, activated NK cells could cause DPSCs to lyse. Furthermore, the expression of activating NK cells' receptors was decreased, but inhibitory receptors of NK cells were elevated following co-cultivation. NK cells acquired CD73 expression, while MSCs could release ATP into the extracellular space where nucleotides were converted into adenosine (ADO) following co-culture system. Under the existence of exogenous 2-chloroadenosine (CADO), the cytotoxic capacity of NK cells was remarkably depressed in a concentration-dependent manner. CONCLUSIONS: DPSCs and BMMSCs could depress NK cells' function by hydrolysing ATP to ADO using CD39 and CD73 enzymatic activity. Our data suggested that DPSCs might represent a new strategy for treating immune-related diseases by regulating previously unrecognized functions in innate immune responses.


Assuntos
Polpa Dentária/citologia , Polpa Dentária/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células-Tronco Mesenquimais/imunologia , 2-Cloroadenosina/farmacologia , 5'-Nucleotidase/metabolismo , Apoptose , Proliferação de Células , Técnicas de Cocultura , Citotoxicidade Imunológica/efeitos dos fármacos , Proteínas Ligadas por GPI/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Células K562 , Células Matadoras Naturais/citologia , Ativação Linfocitária , Purinas/metabolismo , Receptores de Células Matadoras Naturais/efeitos dos fármacos , Receptores de Células Matadoras Naturais/metabolismo , Transdução de Sinais
16.
Artigo em Inglês | MEDLINE | ID: mdl-30925277

RESUMO

Purines and pyrimidines, the important components of DNA and RNA, are closely related to metabolic syndrome and disorder, such as renal disease, gout and diabetic nephropathy etc. Given the importance of the biological significance of purines and pyrimidines, it is necessary to further develop a rapid and sensitive method for practical detection of a large-scale analyses. In this study, based on 96-well solid phase extraction plates-ultra performance liquid chromatography-triple quadrupole mass spectrometry (SPE-UPLC-QqQ-MS/MS), a novel approach for simultaneous determination of 23 purines and pyrimidines in biological samples was developed. First, plasma samples were pretreated by SPE using 96-well plates, which lead to an automated, simplified and rapid sample preparation process. In the methodology development, a large-scale test was performed to evaluate the stability and reliability of the approach. Finally, the levels of purines and pyrimidines in the biological samples were analyzed by this strategy. Experimental results showed that lowest limit of quantification (LLOQ) range from 6.678 × 10-2 µg/mL to 4.275 × 10-6 µg/mL; intra- and inter-day precision are <15% for all analytes. The stability and maximal capability of a single analytical batch could be extended to at least 431 injections (about 70 h). Analysis time of a single run was controlled in 10 min. Under the optimized conditions, wide linear ranges and good correlation coefficients (R2 > 0.99) were acquired. The successful development of this method provides a feasible protocol for a large-scale metabolomics study and it also lays the foundation of quantitative analysis in endogenous analytes.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metabolômica/métodos , Purinas/metabolismo , Pirimidinas/metabolismo , Espectrometria de Massas em Tandem/métodos , Adulto , Animais , Ensaios de Triagem em Larga Escala , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Purinas/sangue , Pirimidinas/sangue , Ratos , Ratos Wistar , Reprodutibilidade dos Testes
17.
PLoS Negl Trop Dis ; 13(3): e0007237, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30870425

RESUMO

Leishmania parasites lack pathways for de novo purine biosynthesis. The depletion of purines induces differentiation into virulent metacyclic forms. In vitro, the parasites can survive prolonged periods of purine withdrawal changing their morphology to long and slender cells with an extended flagellum, and decreasing their translation rates. Reduced translation leads to the appearance of discrete granules that contain LeishIF4E-3, one of the six eIF4E paralogs encoded by the Leishmania genome. We hypothesize that each is responsible for a different function during the life cycle. LeishIF4E-3 is a weak cap-binding protein paralog, but its involvement in translation under normal conditions cannot be excluded. However, in response to nutritional stress, LeishIF4E-3 concentrates in specific cytoplasmic granules. LeishIF4E-3 granulation can be induced by the independent elimination of purines, amino acids and glucose. As these granules contain mature mRNAs, we propose that these bodies store inactive transcripts until recovery from stress occurs. In attempt to examine the content of the nutritional stress-induced granules, they were concentrated over sucrose gradients and further pulled-down by targeting in vivo tagged LeishIF4E-3. Proteomic analysis highlighted granule enrichment with multiple ribosomal proteins, suggesting that ribosome particles are abundant in these foci, as expected in case of translation inhibition. RNA-binding proteins, RNA helicases and metabolic enzymes were also enriched in the granules, whereas no degradation enzymes or P-body markers were detected. The starvation-induced LeishIF4E-3-containing granules, therefore, appear to store stalled ribosomes and ribosomal subunits, along with their associated mRNAs. Following nutritional stress, LeishIF4E-3 becomes phosphorylated at position S75, located in its less-conserved N-terminal extension. The ability of the S75A mutant to form granules was reduced, indicating that cellular signaling regulates LeishIF4E-3 function.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Leishmania/fisiologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Aminoácidos/metabolismo , Grânulos Citoplasmáticos/química , Glucose/metabolismo , Leishmania/metabolismo , Transporte Proteico , Proteoma/análise , Proteínas de Protozoários/análise , Purinas/metabolismo
18.
Thromb Res ; 177: 136-144, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30901608

RESUMO

BACKGROUND: Thrombolytic therapy is effective in fresh deep vein thrombosis (DVT) although the benefit may fall below the risk of bleeding in non-fresh thrombosis. Markers reflecting fresh DVT have not been established. The present study aims to identify metabolites reflecting fresh venous thrombus and their role in thrombus formation. METHODS: Metabolites of rabbit venous blood and jugular venous thrombus 4 h after thrombus induction were analysed using electrophoresis-time of flight mass spectrometry. The effects of the altered metabolites on blood coagulation and platelet aggregation were assessed by using rotation thromboelastometry and platelet aggregometer. Cellular contents and glucose transporter (Glut)-1 expression in aspirated human DVT samples were pathologically analysed. RESULTS: Metabolome analysis identified 226 metabolites (133 cationic and 93 anionic metabolites). Largely altered 18 metabolites (thrombus/blood ratio: >5 or <0.5) included glycolytic metabolites, redox-related metabolites, purine nucleotides and tryptophan metabolites. Among the metabolites with >5-fold increase, lactic acid was most abundant and guanine modestly enhanced whole blood clotting with thromboelastometry. Lactic acid and adenosine monophosphate inhibited collagen-induced platelet aggregation. Human DVTs were rich in erythrocytes expressing Glut-1. The erythrocyte content and Glut-1 expression were negatively correlated with the time after onset of DVT. CONCLUSIONS: Glycolysis-, purine-, and redox-related metabolites may reflect fresh erythrocyte-rich venous thrombus, and altered metabolites may affect venous thrombus formation. An increased level of lactate may reflect active glycolysis of thrombus cellular components, predominantly erythrocytes.


Assuntos
Eritrócitos/metabolismo , Ácido Láctico/metabolismo , Purinas/metabolismo , Trombose Venosa/metabolismo , Animais , Coagulação Sanguínea , Eritrócitos/patologia , Glicólise , Humanos , Ácido Láctico/sangue , Metaboloma , Agregação Plaquetária , Purinas/sangue , Coelhos , Trombose Venosa/sangue , Trombose Venosa/patologia
19.
Leukemia ; 33(4): 815-825, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30846866

RESUMO

The mechanisms that regulate egress of hematopoietic stem/progenitor cells (HSPCs) into peripheral blood (PB) in response to stress, inflammation, tissue/organ injury, or administration of mobilization-inducing drugs are still not well understood, and because of the importance of stem cell trafficking in maintaining organism homeostasis, several complementary pathways are believed to be involved. Our group proposes that mobilization of HSPCs is mainly a result of sterile inflammation in the bone marrow (BM) microenvironment in response to pro-mobilizing stimuli and that during the initiation phase of the mobilization process BM-residing cells belonging to the innate immunity system, including granulocytes and monocytes, release danger-associated molecular pattern molecules (DAMPs, also known as alarmins), reactive oxygen species (ROS), as well as proteolytic and lipolytic enzymes. These factors together orchestrate the release of HSPCs into PB. One of the most important DAMPs released in the initiation phase of mobilization is extracellular adenosine triphosphate, a potent activator of the inflammasome. As a result of its activation, IL-1ß and IL-18 as well as other pro-mobilizing mediators, including DAMPs such as high molecular group box 1 (Hmgb1) and S100 calcium-binding protein A9 (S100a9), are released. These DAMPs are important activators of the complement cascade (ComC) in the mannan-binding lectin (MBL)-dependent pathway. Specifically, Hmgb1 and S100a9 bind to MBL, which leads to activation of MBL-associated proteases, which activate the ComC and in parallel also trigger activation of the coagulation cascade (CoaC). In this review, we will highlight the novel role of the innate immunity cell-expressed NLRP3 inflammasome, which, during the initiation phase of HSPC mobilization, couples purinergic signaling with the MBL-dependent pathway of the ComC and, in parallel, the CoaC for optimal release of HSPCs. These data are important to optimize the pharmacological mobilization of HSPCs.


Assuntos
Medula Óssea/imunologia , Ativação do Complemento/imunologia , Imunidade Inata/imunologia , Inflamassomos/imunologia , Inflamação/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Purinas/metabolismo , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Mobilização de Células-Tronco Hematopoéticas , Humanos , Inflamassomos/metabolismo , Inflamação/patologia
20.
Mycopathologia ; 184(2): 273-281, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30707338

RESUMO

The fertilizing properties of bird manure, or guano, have played an important role in plant cultivation for thousands of years. Research into its chemical composition by Unger in 1846 identified a novel compound, now known as guanine, a purine base that is essential for DNA and RNA biosynthesis and cell signalling. Nitrogen-rich guano can also harbour human pathogens, one significant example being the fungal pathogen Cryptococcus neoformans. Historically associated with pigeon droppings, C. neoformans is able to infect immunocompromised individuals with the aid of a number of adaptive virulence traits. To gain insight into this niche, a quantitative analysis of pigeon guano was performed by LC/MS to determine the concentrations of purines present. Guanine was found in abundance, in particular, in aged guano samples that contained 156-296 µg/g [w/w] compared to 75 µg/g in fresh guano. Adenine concentrations were more consistent between fresh and aged samples, 13 µg/g compared to 10-15 µg/g, respectively. C. neoformans strains that lack key enzymes of the de novo purine synthesis pathway and are guanine or adenine auxotrophs displayed differences in their ability to exploit this substrate: growth of a guanine auxotrophic mutant (gua1Δ) was partially restored on 30% pigeon guano media, but an adenine auxotrophic mutant (ade13Δ) was unable to grow. We conclude that while purine salvage is likely a useful resource-saving mechanism, alone it is not sufficient to fully provide the purines required by wild-type C. neoformans growing in its guano niche.


Assuntos
Columbidae , Cryptococcus neoformans/crescimento & desenvolvimento , Fezes/química , Purinas/análise , Animais , Cromatografia Líquida , Cryptococcus neoformans/metabolismo , Espectrometria de Massas , Viabilidade Microbiana , Purinas/metabolismo
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