RESUMO
The clinically used antihypertensive agent hydralazine rapidly generates hydrazone-derived adducts by reaction with apurinic/apyrimidinic (also known as abasic or AP) sites in many different sequences of duplex DNA. The reaction rates are comparable to those of some AP-trapping reagents previously described as "ultrafast." Initially, reversible formation of a hydrazone adduct is followed by an oxidative cyclization reaction that generates a chemically stable triazolo[3,4-a]phthalazine adduct. The net result is that the reaction of hydralazine with AP sites in duplex DNA yields a rapid and irreversible adduct formation. Although the hydrazone and triazolo[3,4-a]phthalazine adducts differ by only two mass units, it was possible to use MALDI-TOF-MS and ESI-QTOF-nanospray-MS to quantitatively characterize mixtures of these adducts by deconvolution of overlapping isotope envelopes. Reactions of hydralazine with the endogenous ketone pyruvate do not prevent the formation of the hydralazine-AP adducts, providing further evidence that these adducts have the potential to form in cellular DNA. AP sites are ubiquitous in cellular DNA, and rapid, irreversible adduct formation by hydralazine could be relevant to the pathogenesis of systemic drug-induced lupus erythematosus experienced by some patients. Finally, hydralazine might be developed as a probe for the detection of AP sites, the study of cellular BER, and marking the location of AP sites in DNA-sequencing analyses.
Assuntos
Adutos de DNA , DNA , Hidralazina , Ftalazinas , Hidralazina/química , DNA/química , DNA/efeitos dos fármacos , Adutos de DNA/química , Ftalazinas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Anti-Hipertensivos/química , Triazóis/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The reaction of 1,2-aminothiol groups with aldehyde residues in aqueous solution generates thiazolidine products, and this process has been developed as a catalyst-free click reaction for bioconjugation. The work reported here characterized reactions of the biologically relevant 1,2-aminothiols including cysteamine, cysteine methyl ester, and peptides containing N-terminal cysteine residues with the aldehyde residue of apurinic/apyrimidinic (AP) sites in DNA oligomers. These 1,2-aminothiol-containing compounds rapidly generated adducts with AP sites in single-stranded and double-stranded DNA. NMR and MALDI-TOF-MS analyses provided evidence that the reaction generated a thiazolidine product. Conversion of an AP site to a thiazolidine-AP adduct protected against the rapid cleavage normally induced at AP sites by the endonuclease action of the enzyme APE1 and the AP-lyase activity of the biogenic amine spermine. In the presence of excess 1,2-aminothiols, the thiazolidine-AP adducts underwent slow strand cleavage via a ß-lyase reaction that generated products with 1,2-aminothiol-modified sugar residues on the 3'-end of the strand break. In the absence of excess 1,2-aminothiols, the thiazolidine-AP adducts dissociated to release the parent AP-containing oligonucleotide. The properties of the thiazolidine-AP adducts described here mirror critical properties of SRAP proteins HMCES and YedK that capture AP sites in single-stranded regions of cellular DNA and protect them from cleavage.
Assuntos
Cisteína/análogos & derivados , Adutos de DNA , Cisteamina , Reparo do DNA , Tiazolidinas/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/química , Peptídeos , Aldeídos , Dano ao DNARESUMO
Placental trophoblast cells are potentially at risk from circulating endocrine-disrupting chemicals, such as bisphenol A (BPA). To understand how BPA and the reputedly more inert bisphenol S (BPS) affect the placenta, C57BL6J mouse dams were fed 200 µg/kg body weight BPA or BPS daily for 2 wk and then bred. They continued to receive these chemicals until embryonic day 12.5, whereupon placental samples were collected and compared with unexposed controls. BPA and BPS altered the expression of an identical set of 13 genes. Both exposures led to a decrease in the area occupied by spongiotrophoblast relative to trophoblast giant cells (GCs) within the junctional zone, markedly reduced placental serotonin (5-HT) concentrations, and lowered 5-HT GC immunoreactivity. Concentrations of dopamine and 5-hydroxyindoleacetic acid, the main metabolite of serotonin, were increased. GC dopamine immunoreactivity was increased in BPA- and BPS-exposed placentas. A strong positive correlation between 5-HT+ GCs and reductions in spongiotrophoblast to GC area suggests that this neurotransmitter is essential for maintaining cells within the junctional zone. In contrast, a negative correlation existed between dopamine+ GCs and reductions in spongiotrophoblast to GC area ratio. These outcomes lead to the following conclusions. First, BPS exposure causes almost identical placental effects as BPA. Second, a major target of BPA/BPS is either spongiotrophoblast or GCs within the junctional zone. Third, imbalances in neurotransmitter-positive GCs and an observed decrease in docosahexaenoic acid and estradiol, also occurring in response to BPA/BPS exposure, likely affect the placental-brain axis of the developing mouse fetus.
Assuntos
Compostos Benzidrílicos/toxicidade , Encéfalo/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Sulfonas/toxicidade , Trofoblastos/efeitos dos fármacos , Animais , Dopamina/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Serotonina/metabolismo , Trofoblastos/metabolismoRESUMO
Solid-phase microextraction (SPME) was coupled to gas chromatography mass spectrometry (GC-MS) and a method optimized to quantitatively and qualitatively measure a large array of volatile metabolites in alfalfa glandular trichomes isolated from stems, trichome-free stems, and leaves as part of a non-targeted metabolomics approach. Major SPME extraction parameters optimized included SPME fiber composition, extraction temperature, and extraction time. The optimized SPME method provided the most chemically diverse coverage of alfalfa volatile and semi-volatile metabolites using a DVB/CAR/PDMS fiber, extraction temperature of 60 °C, and an extraction time of 20 min. Alfalfa SPME-GC-MS profiles were processed using automated peak deconvolution and identification (AMDIS) and quantitative data extraction software (MET-IDEA). A total of 87 trichome, 59 stem, and 99 leaf volatile metabolites were detected after background subtraction which removed contaminants present in ambient air and associated with the fibers and NaOH/EDTA buffer solution containing CaCl2. Thirty-seven volatile metabolites were detected in all samples, while 15 volatile metabolites were uniquely detected only in glandular trichomes, 9 only in stems, and 33 specifically in leaves as tissue specific volatile metabolites. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of glandular trichomes, stems, and leaves showed that the volatile metabolic profiles obtained from the optimized SPME-GC-MS method clearly differentiated the three tissues (glandular trichomes, stems, and leaves), and the biochemical basis for this differentiation is discussed. Although optimized using plant tissues, the method can be applied to other types of samples including fruits and other foods.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Medicago sativa/química , Metaboloma , Metabolômica , Microextração em Fase Sólida , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/isolamento & purificação , Biologia Computacional/métodos , Análise de Dados , Cromatografia Gasosa-Espectrometria de Massas/métodos , Metabolômica/métodos , Análise de Componente Principal , Microextração em Fase Sólida/métodos , Temperatura , Compostos Orgânicos Voláteis/químicaRESUMO
Azospirillum brasilense is a diazotrophic microorganism capable of associating with roots of important grasses and cereals, promoting plant growth and increasing crop yields. Nitrogen levels and the Ntr regulatory system control the nitrogen metabolism in A. brasilense. This system comprises the nitrogen regulatory proteins GlnD, which is capable of adding uridylyl groups to the PII proteins, GlnB (PII-1) and GlnZ (PII-2), under limiting nitrogen levels. Under such conditions, the histidine kinase NtrB (nitrogen regulatory protein B) cannot interact with GlnB and phosphorylate NtrC (nitrogen regulatory protein C). The phosphorylated form of NtrC acts as a transcriptional activator of genes involved in the metabolism of alternative nitrogen sources. Considering the key role of NtrC in nitrogen metabolism in A. brasilense, in this work we evaluated the proteomic and metabolomic profiles of the wild-type FP2 strain and its mutant ntrC grown under high and low nitrogen. Analysis of the integrated data identifies novel NtrC targets, including proteins involved in the response against oxidative stress (i.e., glutathione S-transferase and hydroperoxide resistance protein), underlining the importance of NtrC to bacterial survival under oxidative stress conditions.
Assuntos
Azospirillum brasilense , Proteômica , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrogênio/metabolismo , Fixação de Nitrogênio , Proteínas PII Reguladoras de Nitrogênio/genética , Proteínas PII Reguladoras de Nitrogênio/metabolismoRESUMO
BACKGROUND: Colorectal cancer (CRC) is a multifactorial disease resulting from both genetic predisposition and environmental factors including the gut microbiota (GM), but deciphering the influence of genetic variants, environmental variables, and interactions with the GM is exceedingly difficult. We previously observed significant differences in intestinal adenoma multiplicity between C57BL/6 J-ApcMin (B6-Min/J) from The Jackson Laboratory (JAX), and original founder strain C57BL/6JD-ApcMin (B6-Min/D) from the University of Wisconsin. METHODS: To resolve genetic and environmental interactions and determine their contributions we utilized two genetically inbred, independently isolated ApcMin mouse colonies that have been separated for over 20 generations. Whole genome sequencing was used to identify genetic variants unique to the two substrains. To determine the influence of genetic variants and the impact of differences in the GM on phenotypic variability, we used complex microbiota targeted rederivation to generate two Apc mutant mouse colonies harboring complex GMs from two different sources (GMJAX originally from JAX or GMHSD originally from Envigo), creating four ApcMin groups. Untargeted metabolomics were used to characterize shifts in the fecal metabolite profile based on genetic variation and differences in the GM. RESULTS: WGS revealed several thousand high quality variants unique to the two substrains. No homozygous variants were present in coding regions, with the vast majority of variants residing in noncoding regions. Host genetic divergence between Min/J and Min/D and the complex GM additively determined differential adenoma susceptibility. Untargeted metabolomics revealed that both genetic lineage and the GM collectively determined the fecal metabolite profile, and that each differentially regulates bile acid (BA) metabolism. Metabolomics pathway analysis facilitated identification of a functionally relevant private noncoding variant associated with the bile acid transporter Fatty acid binding protein 6 (Fabp6). Expression studies demonstrated differential expression of Fabp6 between Min/J and Min/D, and the variant correlates with adenoma multiplicity in backcrossed mice. CONCLUSIONS: We found that both genetic variation and differences in microbiota influences the quantitiative adenoma phenotype in ApcMin mice. These findings demonstrate how the use of metabolomics datasets can aid as a functional genomic tool, and furthermore illustrate the power of a multi-omics approach to dissect complex disease susceptibility of noncoding variants.
Assuntos
Adenoma/genética , Neoplasias Colorretais/genética , Microbioma Gastrointestinal/fisiologia , Predisposição Genética para Doença , Adenoma/metabolismo , Adenoma/microbiologia , Proteína da Polipose Adenomatosa do Colo/genética , Alelos , Animais , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/microbiologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Metabolômica , Metagenômica , Camundongos , MutaçãoRESUMO
12-hydroxy-jasmonoyl-isoleucine (12OH-JA-Ile) is a metabolite in the catabolic pathway of the plant hormone jasmonate, and is synthesized by the cytochrome P450 subclade 94 enzymes. Contrary to the well-established function of jasmonoyl-isoleucine (JA-Ile) as the endogenous bioactive form of jasmonate, the function of 12OH-JA-Ile is unclear. Here, the potential role of 12OH-JA-Ile in jasmonate signaling and wound response was investigated. Exogenous application of 12OH-JA-Ile mimicked several JA-Ile effects including marker gene expression, anthocyanin accumulation and trichome induction in Arabidopsis thaliana. Genome-wide transcriptomics and untargeted metabolite analyses showed large overlaps between those affected by 12OH-JA-Ile and JA-Ile. 12OH-JA-Ile signaling was blocked by mutation in CORONATINE INSENSITIVE 1. Increased anthocyanin accumulation by 12OH-JA-Ile was additionally observed in tomato and sorghum, and was disrupted by the COI1 defect in tomato jai1 mutant. In silico ligand docking predicted that 12OH-JA-Ile can maintain many of the key interactions with COI1-JAZ1 residues identified earlier by crystal structure studies using JA-Ile as ligand. Genetic alternation of jasmonate metabolic pathways in Arabidopsis to deplete both JA-Ile and 12OH-JA-Ile displayed enhanced jasmonate deficient wound phenotypes and was more susceptible to insect herbivory than that depleted in only JA-Ile. Conversely, mutants overaccumulating 12OH-JA-Ile showed intensified wound responses compared with wild type with similar JA-Ile content. These data are indicative of 12OH-JA-Ile functioning as an active jasmonate signal and contributing to wound and defense response in higher plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Isoleucina/análogos & derivados , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Isoleucina/metabolismo , Redes e Vias Metabólicas , Fenótipo , Transdução de SinaisRESUMO
Paternal environment can induce detrimental developmental origins of health and disease (DOHaD) effects in resulting offspring and even future descendants. Such paternal-induced DOHaD effects might originate from alterations in a possible seminal fluid microbiome (SFM) and composite metabolome. Seminal vesicles secrete a slightly basic product enriched with fructose and other carbohydrates, providing an ideal habitat for microorganisms. Past studies confirm the existence of a SFM that is influenced by genetic and nutritional status. Herein, we sought to determine whether treatment of male mice with a combination of antibiotics designed to target SFM induces metabolic alterations in seminal vesicle gland secretions (seminal fluid) and histopathological changes in testes and epididymides. Adult (10- to 12-week-old) National Institutes of Health (NIH) Swiss males (n = 10 per group) were treated with Clindamycin 0.06 mg/kg day, Unasyn (ampicillin/sulbactam) 40 mg/kg day and Baytril (enrofloxacin) 50 mg/kg day designed to target the primary bacteria within the SFM or saline vehicle alone. Fourteen-day antibiotic treatment of males induced metabolomic changes in seminal vesicles with inosine, xanthine and l-glutamic acid decreased but d-fructose increased in glandular secretions. While spermatogenesis was not affected in treated males, increased number of epididymal tubules showed cribriform growth in this group (7 antibiotic-treated males: 3 saline control males; P = 0.01). Antibiotic-treated males showed more severe cribriform cysts. Current findings suggest antibiotic treatment of male mice results in seminal fluid metabolome and epididymal histopathological alterations. It remains to be determined whether such changes compromise male reproductive function or lead to DOHaD effects in resulting offspring.
Assuntos
Antibacterianos/farmacologia , Epididimo/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Metaboloma/efeitos dos fármacos , Sêmen/efeitos dos fármacos , Ampicilina/farmacologia , Animais , Enrofloxacina , Epididimo/metabolismo , Masculino , Camundongos , Sêmen/metabolismo , Sulbactam/farmacologia , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
Custom software entitled Plant Metabolite Annotation Toolbox (PlantMAT) has been developed to address the number one grand challenge in metabolomics, which is the large-scale and confident identification of metabolites. PlantMAT uses informed phytochemical knowledge for the prediction of plant natural products such as saponins and glycosylated flavonoids through combinatorial enumeration of aglycone, glycosyl, and acyl subunits. Many of the predicted structures have yet to be characterized and are absent from traditional chemical databases, but have a higher probability of being present in planta. PlantMAT allows users to operate an automated and streamlined workflow for metabolite annotation from a user-friendly interface within Microsoft Excel, a familiar, easily accessed program for chemists and biologists. The usefulness of PlantMAT is exemplified using ultrahigh-performance liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-QTOF-MS/MS) metabolite profiling data of saponins and glycosylated flavonoids from the model legume Medicago truncatula. The results demonstrate PlantMAT substantially increases the chemical/metabolic space of traditional chemical databases. Ten of the PlantMAT-predicted identifications were validated and confirmed through the isolation of the compounds using ultrahigh-performance liquid chromatography-mass spectrometry-solid-phase extraction (UHPLC-MS-SPE) followed by de novo structural elucidation using 1D/2D nuclear magnetic resonance (NMR). It is further demonstrated that PlantMAT enables the dereplication of previously identified metabolites and is also a powerful tool for the discovery of structurally novel metabolites.
Assuntos
Flavonoides/metabolismo , Medicago truncatula/metabolismo , Metabolômica , Extratos Vegetais/metabolismo , Saponinas/metabolismo , Software , Cromatografia Líquida de Alta Pressão , Flavonoides/análise , Glicosilação , Medicago truncatula/química , Extratos Vegetais/análise , Saponinas/análise , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Plant metabolomics has matured and modern plant metabolomics has accelerated gene discoveries and the elucidation of a variety of plant natural product biosynthetic pathways. This review covers the approximate period of 2000 to 2014, and highlights specific examples of the discovery and characterization of novel genes and enzymes associated with the biosynthesis of natural products such as flavonoids, glucosinolates, terpenoids, and alkaloids. Additional examples of the integration of metabolomics with genome-based functional characterizations of plant natural products that are important to modern pharmaceutical technology are also reviewed. This article also provides a substantial review of recent technical advances in mass spectrometry imaging, nuclear magnetic resonance imaging, integrated LC-MS-SPE-NMR for metabolite identifications, and X-ray crystallography of microgram quantities for structural determinations. The review closes with a discussion on the future prospects of metabolomics related to crop species and herbal medicine.
Assuntos
Produtos Biológicos , Metabolômica , Plantas , Alcaloides/química , Alcaloides/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Cristalografia por Raios X , Flavonoides/química , Flavonoides/isolamento & purificação , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Plantas/química , Plantas/enzimologia , Plantas/genética , Terpenos/química , Terpenos/isolamento & purificaçãoRESUMO
Liquid chromatography/mass spectrometry (LC/MS) metabolite profiling has been widely used in comparative metabolomics studies; however, LC/MS-based comparative metabolomics currently faces several critical challenges. One of the greatest challenges is how to effectively align metabolites across different LC/MS profiles; a single metabolite can give rise to multiple peak features, and the grouped peak features that can be used to construct a spectrum pattern of single metabolite can vary greatly between biochemical experiments and even between instrument runs. Another major challenge is that the observed retention time for a single metabolite can also be significantly affected by experimental conditions. To overcome these two key challenges, we present a novel metabolite-based alignment approach entitled MET-XAlign to align metabolites across LC/MS metabolomics profiles. MET-XAlign takes the deduced molecular mass and estimated compound retention time information that can be extracted by our previously published tool, MET-COFEA, and aligns metabolites based on this information. We demonstrate that MET-XAlign is able to cross-align metabolite compounds, either known or unknown, in LC/MS profiles not only across different samples but also across different biological experiments and different electrospray ionization modes. Therefore, our proposed metabolite-based cross-alignment approach is a great step forward and its implementation, MET-XAlign, is a very useful tool in LC/MS-based comparative metabolomics. MET-XAlign has been successfully implemented with core algorithm coding in C++, making it very efficient, and visualization interface coding in the Microsoft.NET Framework. The MET-XAlign software along with demonstrative data is freely available at http://bioinfo.noble.org/manuscript-support/met-xalign/ .
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Estatística como Assunto/métodos , Biomarcadores/metabolismoRESUMO
A plant natural product tandem mass spectral library has been constructed using authentic standards and purified compounds. Currently, the library contains 1734 tandem mass spectra for 289 compounds, with the majority (76%) of the compounds being plant phenolics such as flavonoids, isoflavonoids, and phenylpropanoids. Tandem mass spectra and chromatographic retention data were acquired on a triple quadrupole mass spectrometer coupled to an ultrahigh pressure liquid chromatograph using six different collision energies (CEs) (10-60 eV). Comparative analyses of the tandem mass spectral data revealed that the loss of ring substituents preceded the C-ring opening during the fragmentation of flavonoids and isoflavonoids. At lower CE (i.e., 10 and 20 eV), the flavonoids and isoflavonoid central ring structures typically remained intact, and fragmentation was characterized by the loss of the substituents (i.e., methyl and glycosyl groups). At higher CE, the flavonoid and isoflavonoid core ring systems underwent C-ring cleavage and/or rearrangement depending on the structure, particularly hydroxylation patterns. In-source electrochemical oxidation was observed for phenolics that had ortho-diphenol moieties (i.e., vicinal hydroxyl groups on the aromatic rings). The ortho-diphenols were oxidized to ortho-quinones, yielding an intensive and, in most cases, a base ion peak corresponding to a [(M - 2H) - H](-) ion in their mass spectra. The library also contains reverse-phase retention times, allowing for the construction, validation, and testing of an artificial neural network retention prediction of other flavonoids and isoflavonoids not contained within the library. The library is freely available for nonprofit, academic use and it can be downloaded at http://www.noble.org/apps/Scientific/WebDownloadManager/DownloadArea.aspx.
Assuntos
Produtos Biológicos/química , Bases de Dados de Compostos Químicos , Fenilpropionatos/isolamento & purificação , Extratos Vegetais/química , Produtos Biológicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Flavonoides/química , Flavonoides/isolamento & purificação , Disseminação de Informação , Internet , Isoflavonas/química , Isoflavonas/isolamento & purificação , Estrutura Molecular , Fenilpropionatos/química , Espectrometria de Massas em TandemRESUMO
In this paper, we present a novel liquid chromatography/mass spectrometry (LC/MS) data processing and analysis platform, MET-COFEA (METabolite COmpound Feature Extraction and Annotation). MET-COFEA detects and clusters chromatographic peak features for each metabolite compound by first comprehensively evaluating retention time and peak shape criteria and then annotating the associations between each peak's observed m/z value with the corresponding metabolite compound's molecular mass. MET-COFEA integrates a series of innovative approaches, including novel mass trace based extracted-ion chromatogram (EIC) extraction, continuous wavelet transform (CWT)-based peak detection, and compound-associated peak clustering and peak annotation algorithms. On the basis of the deduced neutral molecular mass and retention time, we have also developed a new alignment algorithm that uses compound-associated peak groups instead of individual peaks to align the same metabolite compound across samples from different electrospray ionization (ESI) modes, different instruments, even different experimental conditions. MET-COFEA has been systematically tested on a series of LC/MS profiles of mixed standards at different concentrations as well as real untargeted LC/MS plant metabolomics data. We compared the performances of MET-COFEA with the existing publicly available tools at LC/MS peak analysis level and demonstrated its excellent performance in this arena. MET-COFEA is freely available at http://bioinfo.noble.org/manuscript-support/met-cofea/.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Software , AlgoritmosRESUMO
Tobacco mosaic virus (TMV) forms dense cytoplasmic bodies containing replication-associated proteins (virus replication complexes [VRCs]) upon infection. To identify host proteins that interact with individual viral components of VRCs or VRCs in toto, we isolated viral replicase- and VRC-enriched fractions from TMV-infected Nicotiana tabacum plants. Two host proteins in enriched fractions, ATP-synthase γ-subunit (AtpC) and Rubisco activase (RCA) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry. Through pull-down analysis, RCA bound predominantly to the region between the methyltransferase and helicase domains of the TMV replicase. Tobamovirus, but not Cucumber mosaic virus or Potato virus X, infection of N. tabacum plants resulted in 50% reductions in Rca and AtpC messenger RNA levels. To investigate the role of these host proteins in TMV accumulation and plant defense, we used a Tobacco rattle virus vector to silence these genes in Nicotiana benthamiana plants prior to challenge with TMV expressing green fluorescent protein. TMV-induced fluorescent lesions on Rca- or AtpC-silenced leaves were, respectively, similar or twice the size of those on leaves expressing these genes. Silencing Rca and AtpC did not influence the spread of Tomato bushy stunt virus and Potato virus X. In AtpC- and Rca-silenced leaves TMV accumulation and pathogenicity were greatly enhanced, suggesting a role of both host-encoded proteins in a defense response against TMV. In addition, silencing these host genes altered the phenotype of the TMV infection foci and VRCs, yielding foci with concentric fluorescent rings and dramatically more but smaller VRCs. The concentric rings occurred through renewed virus accumulation internal to the infection front.
Assuntos
Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Nicotiana/virologia , Vírus do Mosaico do Tabaco/fisiologia , Proteínas de Cloroplastos/genética , Cloroplastos/virologia , Inativação Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno , Fenótipo , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potexvirus/metabolismo , Potexvirus/patogenicidade , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Nicotiana/genética , Nicotiana/metabolismo , Vírus do Mosaico do Tabaco/enzimologia , Vírus do Mosaico do Tabaco/patogenicidade , Tombusvirus/metabolismo , Tombusvirus/patogenicidade , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
SCOPE: Obesity and its metabolic comorbidities pose a major global challenge for public health. Glucoraphanin (GRN) is a natural bioactive compound enriched in broccoli that is known to have potential health benefits against various human chronic diseases. METHODS AND RESULTS: This study investigats the effects of broccoli GRN supplementation on body weight, metabolic parameters, gut microbiome and metabolome associated with obesity. The study is conducted on an obese-related C57BL/6J mouse model through the treatment of normal control diet, high-fat diet (HFD)and GRN-supplemented HFD (HFD-GRN) to determine the metabolic protection of GRN. The results shows that GRN treatment alleviates obesity-related traits leading to improved glucose metabolism in HFD-fed animals. Mechanically, the study noticed that GRN significantly shifts the gut microbial diversity and composition to an eubiosis status. GRN supplement also significantly alters plasma metabolite profiles. Further integrated analysis reveal a complex interaction between the gut microbes and host metabolism that may contribute to GRN-induced beneficial effects against HFD. CONCLUSION: These results indicate that beneficial effects of broccoli GRN on reversing HFD-induced adverse metabolic parameters may be attributed to its impacts on reprogramming microbial community and metabolites. Identification of the mechanistic functions of GRN further warrants it as a dietary candidate for obesity prevention.
Assuntos
Brassica , Dieta Hiperlipídica , Suplementos Nutricionais , Microbioma Gastrointestinal , Glucosinolatos , Imidoésteres , Metaboloma , Camundongos Endogâmicos C57BL , Obesidade , Oximas , Sulfóxidos , Microbioma Gastrointestinal/efeitos dos fármacos , Animais , Obesidade/microbiologia , Obesidade/tratamento farmacológico , Dieta Hiperlipídica/efeitos adversos , Brassica/química , Glucosinolatos/farmacologia , Masculino , Metaboloma/efeitos dos fármacos , Sulfóxidos/farmacologia , Imidoésteres/farmacologia , Oximas/farmacologia , CamundongosRESUMO
LC-MS/MS analyses have been reported as challenging for the reliable separation and quantification of cyanogenic glycosides (CNGs), especially (R)-prunasin and sambunigrin isomers found in American elderberry (Sambucus nigra L. subsp. canadensis (L.) Bolli). Hence, a novel multiple reaction monitoring (MRM)-based ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated in the present study for simultaneous separation and quantification of five CNGs, including amygdalin, dhurrin, linamarin, (R)-prunasin, and (S)-prunasin (commonly referred to as sambunigrin). Initially, the role of ammonium formate was investigated as an aqueous mobile-phase additive in developing MRM-based UHPLC-MS/MS. Later, chromatographic conditions for the resolved separation of (R)-prunasin and sambunigrin were identified. Validation studies confirmed that the developed method has good linearity and acceptable precision and accuracy. A noticeable matrix effect (mainly signal enhancement) was observed in leaf samples only. This method was used to detect and quantify CNGs, including (R)-prunasin and sambunigrin, in leaf and fruit samples of American elderberry. Among the studied CNGs, only (R)-prunasin was detected in the leaf samples. Interestingly, (S)-prunasin (sambunigrin) was not detected in the samples analyzed, even though it has been previously reported in elderberry species.
RESUMO
Prenylated flavonoids and isoflavonoids possess antimicrobial activity against fungal pathogens of plants. However, only a few plant flavonoid and isoflavonoid prenyltransferase genes have been identified to date. In this study, an isoflavonoid prenyltransferase gene, designated as LaPT1, was identified from white lupin (Lupinus albus). The deduced protein sequence of LaPT1 shared high homologies with known flavonoid and isoflavonoid prenyltransferases. The LaPT1 gene was mainly expressed in roots, a major site for constitutive accumulation of prenylated isoflavones in white lupin. LaPT1 is predicted to be a membrane-bound protein with nine transmembrane regions and conserved functional domains similar to other flavonoid and isoflavonoid prenyltransferases; it has a predicted chloroplast transit peptide and is plastid localized. A microsomal fraction containing recombinant LaPT1 prenylated the isoflavone genistein at the B-ring 3' position to produce isowighteone. The enzyme is also active with 2'-hydroxygenistein but has no activity with other flavonoid substrates. The apparent K(m) of recombinant LaPT1 for the dimethylallyl diphosphate prenyl donor is in a similar range to that of other flavonoid prenyltransferases, but the apparent catalytic efficiency with genistein is considerably higher. Removal of the transit peptide increased the apparent overall activity but also increased the K(m). Medicago truncatula hairy roots expressing LaPT1 accumulated isowighteone, a compound that is not naturally produced in this species, indicating a strategy for metabolic engineering of novel antimicrobial compounds in legumes.
Assuntos
Dimetilaliltranstransferase/metabolismo , Lupinus/enzimologia , Proteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , DNA Complementar/metabolismo , Dimetilaliltranstransferase/genética , Ativação Enzimática , Perfilação da Expressão Gênica , Genes de Plantas , Genisteína/isolamento & purificação , Genisteína/metabolismo , Lupinus/genética , Medicago truncatula/enzimologia , Medicago truncatula/genética , Proteínas de Membrana/genética , Engenharia Metabólica , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plastídeos/enzimologia , Plastídeos/genética , Prenilação , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Colon cancer onset is strongly associated with the differences in microbial taxa in the gastrointestinal tract. Although recent studies highlight the role of individual taxa, the effect of a complex gut microbiome (GM) on the metabolome and host transcriptome is still unknown. We used a multi-omics approach to determine how differences in the GM affect the susceptibility to adenoma development in a rat model of human colon cancer. Ultra-high performance liquid chromatography mass spectrometry of feces collected prior to observable disease onset identified putative metabolite profiles that likely predict future disease severity. Transcriptome analyses performed after disease onset from normal colonic epithelium and tumor tissues show a correlation between GM and host gene expression. Integrated pathway analyses of the metabolome and transcriptome based on putatively identified metabolic features indicate that bile acid biosynthesis is enriched in rats with high tumors along with increased fatty acid metabolism and mucin biosynthesis. Targeted pyrosequencing of the Pirc allele indicates that the GM alters the mechanism of adenoma development and may drive an epigenetic pathway of tumor suppressor silencing. This study reveals how untargeted metabolomics identifies signatures of susceptibility and integrated analyses uncover pathways of differential mechanisms of loss of tumor suppressor gene function and for potential prevention and therapeutic intervention. IMPORTANCE The association between the gut microbiome and colon cancer is significant but difficult to test in model systems. This study highlights the association of differences in the pathogen-free gut microbiome to changes in the host transcriptome and metabolome that correlate with colon adenoma initiation and development in a rat genetic model of early colon cancer. The utilization of a multi-omics approach integrating metabolomics and transcriptomics reveals differences in pathways including bile acid biosynthesis and fatty acid metabolism. The study also shows that differences in gut microbiomes significantly alter the mechanism of adenoma formation, shifting from genetic changes to epigenetic changes that initiate the early loss of tumor suppressor function. These findings enhance our understanding of the gut microbiome's role in colon cancer susceptibility, offer insights into potential biomarkers and therapeutic targets, and may pave the way for future prevention and intervention strategies.
Assuntos
Adenoma , Neoplasias do Colo , Microbioma Gastrointestinal , Humanos , Ratos , Animais , Microbioma Gastrointestinal/genética , Multiômica , Adenoma/genética , Neoplasias do Colo/genética , Ácidos e Sais Biliares , Ácidos GraxosRESUMO
Glansreginin A has been reported to be an indicator of the quality of walnuts (Juglans spp.). However, bioactive properties of glansreginin A have not been adequately explored. In the present study, we quantified concentrations of glansreginin A in black walnuts (Juglans nigra) using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed an array of in vitro bioassays to characterize biological activities (e.g., antibacterial, antioxidant, anticancer capacities) of this compound. Results from HPLC-MS/MS analysis indicated that glansreginin A was presented in all 12 black cultivars examined and its contents were variable among black walnut cultivars, ranged from 6.8 mg/kg (Jackson) to 47.0 mg/kg (Hay). Glansreginin A possessed moderate antibacterial activities against Gram-positive pathogens (Staphylococcus aureus and Bacillus anthracis). This compound exhibited no antioxidant activities, did not induce the activity of antioxidant response element signaling pathways, and exerted no antiproliferative effects on tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells.
Assuntos
Juglans , Quinolinas , Juglans/química , Espectrometria de Massas em Tandem/métodos , Antioxidantes/farmacologia , Antioxidantes/química , Antibacterianos/farmacologiaRESUMO
MS has evolved as a critical component in metabolomics, which seeks to answer biological questions through large-scale qualitative and quantitative analyses of the metabolome. MS-based metabolomics techniques offer an excellent combination of sensitivity and selectivity, and they have become an indispensable platform in biology and metabolomics. In this minireview, various MS technologies used in metabolomics are briefly discussed, and future needs are suggested.