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1.
Food Chem ; 305: 125450, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499291

RESUMO

A round robin comparison was performed in order to test the performance of a recently developed LC-MS/MS method for quantification of 6 folate forms. Eighty-nine samples representing the food groups of fruits, vegetables, legumes, cereals, dairy products, meat, and offal were analyzed by two LC-MS/MS methods and a microbiological assay (MA). A plant-origin deconjugase enzyme (Arabidopsis thaliana) for deconjugation of folates (PE-LC-MS/MS), or animal-origin deconjugase (rat serum and chicken pancreas) (AE-LC-MS/MS) was used in the LC-MS/MS methods, each in a single enzymatic step. In contrast, the MA involved tri-enzyme extraction including human plasma as a deconjugase. A significant bias of 17% lower and 25% higher results was found when PE-LC-MS/MS was compared to MA and AE-LC-MS/MS, respectively. The PE-LC-MS/MS provides fast quantification of various folate vitamers and total folate content, which could be a proper substitute to the currently standardized but imprecise and time-consuming microbiological assay in the future.

2.
Food Chem ; 303: 125363, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31472383

RESUMO

Present in many plant foods, biogenic phenolic compounds are important bioactive phytonutrients with high anti-oxidant activity and thereby are praised for their health-promoting properties. However, current food nutrient improvement by high phenolic content in staples is limited by the shortage of genetic resources rich in phenolic compounds. To resolve this obstacle, we developed a non-destructive massive analytical approach to screen wheat phenolic mutants. In grains, multiple mutant lines showed significantly higher contents of flavonoids or cell wall-bound phenolic esters. Moreover, five mutants showed higher anti-oxidant potentials in wall-bound phenolic compounds ranging from 15% to 20%, with the maximal close to natural black wheat. In contrast to black wheat, two mutants accumulated higher phenolic compounds in the endosperm. lrf4 was mapped by BSR to a concentrated genomic region in the short arm of chromosome 1A. The present work represents an efficient high-throughput strategy to increase wheat anti-oxidant potential through traditional mutagenesis.


Assuntos
Antioxidantes/metabolismo , Mutação , Fenóis/metabolismo , Triticum/genética , Triticum/metabolismo , Flavonoides/metabolismo
3.
Molecules ; 24(13)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31247930

RESUMO

Acetogenins are bioactive fatty acid derivatives found in avocado tissues. Their efficacy as antimicrobials has been documented and initiated interest to use them as replacements of synthetic food additives. The present work focused on evaluation of multiple analytical methodologies for detection and quantification of organic solids present in a food-grade acetogenin-enriched extract (Avosafe®), and on its safety evaluations using bacterial reverse mutation (AMES) tests and acute oral toxicity to rat assays. Results confirmed chemical structures of two acetogenins as present in Avosafe® (AcO-avocadyne-(0) and AcO-avocadiene B-(3)), and together with seven other previously known compounds, quantified 94.74 ± 5.77% w/w of its solids as acetogenins. Safety evaluations indicated that Avosafe® was non-mutagenic and had an acute median lethal oral dose (LD50) to rats higher than the maximum concentration tested (>2000 mg·kg-1), with no signs of macroscopic abnormalities in organs. Mean body weight and hematological and biochemical parameters were normal after 14 days of a single oral dose of 2000 mg·kg-1. The results advance scientific information on the safety of avocado seed acetogenins and also generate new knowledge on profiles and concentrations of individual acetogenins found in avocado tissues (seed, pulp, and leaves) and in Avosafe®.

4.
Food Chem ; 277: 362-372, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30502158

RESUMO

High hydrostatic pressure (HHP) processing is a non-thermal technology reported to increase desirable metabolites in plant foods. This work evaluated changes in carotenoid accumulation in fresh-cut papaya fruit as affected by HHP treatment (50-400 MPa for 3-60 min) and during subsequent storage at 4 °C; simultaneously, transcriptional activities of carotenoid biosynthetic genes and oxidative stress markers were evaluated. LC-MS analyses revealed that HHP treatment increased carotenoid precursors and carotenes contents following processing and storage: lycopene levels increased up to 11-fold compared to the non-treated samples, and H2O2 and lipid peroxidation were concomitantly increased. qRT-PCR of intact RNA showed that the amount of phytoene desaturase transcripts increased after HHP treatment, and that they were correlated with carotene accumulation. This is the first study to show that HHP treatment triggers de novo carotenoid biosynthesis, which is regulated at the transcriptional level, possibly by inducing oxidative stress signaling in fruit tissue.


Assuntos
Carica/metabolismo , Carotenoides/biossíntese , Frutas/metabolismo , Carica/genética , Cromatografia Líquida , Temperatura Baixa , Manipulação de Alimentos , Frutas/genética , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Pressão Hidrostática , Peroxidação de Lipídeos/efeitos dos fármacos , Licopeno/análise , Análise Multivariada , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ativação Transcricional/genética
5.
BMC Plant Biol ; 17(1): 159, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28969589

RESUMO

BACKGROUND: Avocado fruit contains aliphatic acetogenins (oft-acetylated, odd-chain fatty alcohols) with promising bioactivities for both medical and food industries. However, we have scarce knowledge about their metabolism. The present work aimed to study changes in acetogenin profiles from mesocarp, lipid-containing idioblasts, and seeds from 'Hass' cultivar during fruit development, germination, and three harvesting years. An untargeted LC-MS based lipidomic analysis was also conducted to profile the lipidome of avocado fruit in each tissue. RESULTS: The targeted analysis showed that acetogenin profiles and contents remained unchanged in avocado mesocarp during maturation and postharvest ripening, germination, and different harvesting years. However, a shift in the acetogenin profile distribution, accompanied with a sharp increase in concentration, was observed in seed during early maturation. Untargeted lipidomics showed that this shift was accompanied with remodeling of glycerolipids: TAGs and DAGs decreased during fruit growing in seed. Remarkably, the majority of the lipidome in mature seed was composed by acetogenins; we suggest that this tissue is able to synthesize them independently from mesocarp. On the other hand, lipid-containing idioblasts accumulated almost the entire acetogenin pool measured in the whole mesocarp, while only having 4% of the total fatty acids. The lipidome of this cell type changed the most when the fruit was ripening after harvesting, TAGs decreased while odd-chain DAGs increased. Notably, idioblast lipidome was more diverse than that from mesocarp. CONCLUSIONS: Evidence shown here suggests that idioblasts are the main site of acetogenin biosynthesis in avocado mesocarp. This work unveiled the prevalence of aliphatic acetogenins in the avocado fruit lipidome and evidenced TAGs as initial donors of the acetogenin backbones in its biosynthesis. It also sets evidence for acetogenins being included in future works aimed at characterizing the avocado seed, as they are a main component of their lipidome.


Assuntos
Acetogeninas/metabolismo , Frutas/metabolismo , Persea/fisiologia , Frutas/crescimento & desenvolvimento , Germinação , Metabolismo dos Lipídeos , Persea/citologia , Células Vegetais/metabolismo , Sementes
6.
Nat Commun ; 7: 11640, 2016 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-27291711

RESUMO

DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilação de DNA/genética , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Proteínas de Arabidopsis/genética , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Desmetilação do DNA , Epigênese Genética , Ácido Fólico/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Homeostase/efeitos dos fármacos , Lisina/metabolismo , Meteniltetra-Hidrofolato Cicloidrolase/genética , Metionina/farmacologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Modelos Biológicos , Mutação/genética , Transporte Proteico/efeitos dos fármacos , S-Adenosilmetionina/metabolismo , Tetra-Hidrofolatos/farmacologia
7.
Front Microbiol ; 7: 431, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27065985

RESUMO

Tetrahydrofolate (THF) and its one-carbon derivatives, collectively termed folates, are essential cofactors, but are inherently unstable. While it is clear that chemical oxidation can cleave folates or damage their pterin precursors, very little is known about enzymatic damage to these molecules or about whether the folate biosynthesis pathway responds adaptively to damage to its end-products. The presence of a duplication of the gene encoding the folate biosynthesis enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (FolK) in many sequenced bacterial genomes combined with a strong chromosomal clustering of the folK gene with panB, encoding the 5,10-methylene-THF-dependent enzyme ketopantoate hydroxymethyltransferase, led us to infer that PanB has a side activity that cleaves 5,10-methylene-THF, yielding a pterin product that is recycled by FolK. Genetic and metabolic analyses of Escherichia coli strains showed that overexpression of PanB leads to accumulation of the likely folate cleavage product 6-hydroxymethylpterin and other pterins in cells and medium, and-unexpectedly-to a 46% increase in total folate content. In silico modeling of the folate biosynthesis pathway showed that these observations are consistent with the in vivo cleavage of 5,10-methylene-THF by a side-activity of PanB, with FolK-mediated recycling of the pterin cleavage product, and with regulation of folate biosynthesis by folates or their damage products.

8.
New Phytol ; 202(4): 1223-36, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24635769

RESUMO

Roots have both indeterminate and determinate developmental programs. The latter is preceded by the former. It is not well understood how the indeterminacy-to-determinacy switch (IDS) is regulated. We isolated a moots koom2 (mko2; 'short root' in Mayan) Arabidopsis thaliana mutant with determinate primary root growth and analyzed the root apical meristem (RAM) behavior using various marker lines. Deep sequencing and genetic and pharmacological complementation permitted the identification of a point mutation in the FOLYLPOLYGLUTAMATE SYNTHETASE1 (FPGS1) gene responsible for the mko2 phenotype. Wild-type FPGS1 is required to maintain the IDS in the 'off' state. When FPGS1 function is compromised, the IDS is turned on and the RAM becomes completely consumed. The polyglutamate-dependent pathway of the IDS involves activation of the quiescent center independently of auxin gradients and regulatory modules participating in RAM maintenance (WUSCHEL-RELATED HOMEOBOX5 (WOX5), PLETHORA, and SCARECROW (SCR)). The mko2 mutation causes drastic changes in folate metabolism and also affects lateral root primordium morphogenesis but not initiation. We identified a metabolism-dependent pathway involved in the IDS in roots. We suggest that the root IDS represents a specific developmental pathway that regulates RAM behaviour and is a different level of regulation in addition to RAM maintenance.


Assuntos
Arabidopsis/genética , Ácido Fólico/metabolismo , Peptídeo Sintases/genética , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Meristema/citologia , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Peptídeo Sintases/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Mutação Puntual , Transdução de Sinais , Nicho de Células-Tronco
9.
Plant Physiol ; 155(3): 1237-51, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21233333

RESUMO

A recessive Arabidopsis (Arabidopsis thaliana) mutant with short primary roots and root hairs was identified from a forward genetic screen. The disrupted gene in the mutant encoded the plastidial isoform of folylpolyglutamate synthetase (FPGS), previously designated as AtDFB, an enzyme that catalyzes the addition of glutamate residues to the folate molecule to form folylpolyglutamates. The short primary root of atdfb was associated with a disorganized quiescent center, dissipated auxin gradient in the root cap, bundled actin cytoskeleton, and reduced cell division and expansion. The accumulation of monoglutamylated forms of some folate classes in atdfb was consistent with impaired FPGS function. The observed cellular defects in roots of atdfb underscore the essential role of folylpolyglutamates in the highly compartmentalized one-carbon transfer reactions (C1 metabolism) that lead to the biosynthesis of compounds required for metabolically active cells found in the growing root apex. Indeed, metabolic profiling uncovered a depletion of several amino acids and nucleotides in atdfb indicative of broad alterations in metabolism. Methionine and purines, which are synthesized de novo in plastids via C1 enzymatic reactions, were particularly depleted. The root growth and quiescent center defects of atdfb were rescued by exogenous application of 5-formyl-tetrahydrofolate, a stable folate that was readily converted to metabolically active folates. Collectively, our results indicate that AtDFB is the predominant FPGS isoform that generates polyglutamylated folate cofactors to support C1 metabolism required for meristem maintenance and cell expansion during postembryonic root development in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Peptídeo Sintases/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Plastídeos/enzimologia , Arabidopsis/citologia , Arabidopsis/genética , Carbono/metabolismo , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Genoma de Planta/genética , Ácido Glutâmico/metabolismo , Guanosina/farmacologia , Ácidos Indolacéticos/metabolismo , Isoenzimas/metabolismo , Metaboloma/efeitos dos fármacos , Metionina/farmacologia , Mutação/genética , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo , Tetra-Hidrofolatos/metabolismo
10.
J Agric Food Chem ; 59(4): 1095-103, 2011 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-21254774

RESUMO

This study investigated the effects of broccoli sprouts (BS) on sterol and lipid homeostasis in Syrian hamsters with dietary-induced hypercholesterolemia. Treatments included freeze-dried BS containing 2 or 20 µmol of glucoraphanine (BSX, BS10X), glucoraphanine-rich BS extract (GRE), sulforaphane-rich BS extract (SFE), and simvastatin. Each experimental diet was offered to eight animals (male and female) for 7 weeks. Hepatic cholesterol was reduced by BS10X and SFE treatments in all animals. This correlated with a down-regulation of gene expression of sterol regulatory element-binding proteins (SREBP-1 and -2) and fatty acid synthase (FAS) caused by GRE and SFE diets. BS10X caused changes in gene expression in a gender-specific manner; additionally, it increased coprostanol excretion in females. With the same concentration of glucoraphanin, consumption of broccoli sprouts (BS10X) had more marked effects on cholesterol homeostasis than GRE; this finding reinforces the importance of the matrix effects on the bioactivity of functional ingredients.


Assuntos
Brassica/química , Glucosinolatos/análise , Isotiocianatos/análise , Metabolismo dos Lipídeos/genética , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Animais , Anticolesterolemiantes , Colesterol/metabolismo , Cricetinae , Ácido Graxo Sintases/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Masculino , Mesocricetus , Brotos de Planta/química , Fatores Sexuais , Proteínas de Ligação a Elemento Regulador de Esterol/genética
11.
Electrophoresis ; 30(24): 4195-205, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20013902

RESUMO

Electrokinetic techniques offer a great potential for biological particle manipulation. Among these, dielectrophoresis (DEP) has been successfully utilized for the concentration of bioparticles. Traditionally, DEP is performed employing microelectrodes, an approach with attractive characteristics but expensive due to microelectrode fabrication costs. An alternative is insulator-based DEP, a method where non-uniform electric fields are created with arrays of insulating structures. This study presents the concentration of linear DNA particles (pET28b) employing a microchannel, with an array of cylindrical insulating structures and direct current electric fields. Results showed manipulation of DNA particles with a combination of electroosmotic, electrophoretic, and dielectrophoretic forces. Employing suspending media with conductivity of 104 muS/cm and pH of 11.15, under applied fields between 500 and 1500 V/cm, DNA particles were observed to be immobilized due to negative dielectrophoretic trapping. The observation of DNA aggregates that occurred at higher applied fields, and dispersed once the field was removed is also included. Finally, concentration factors varying from 8 to 24 times the feed concentration were measured at 2000 V/cm after concentration time-periods of 20-40 s. The results presented here demonstrate the potential of insulator-based DEP for DNA concentration, and open the possibility for fast DNA manipulation for laboratory and large-scale applications.


Assuntos
DNA/química , Eletroforese/métodos , DNA/isolamento & purificação , Eletroforese/instrumentação , Microfluídica/instrumentação , Microfluídica/métodos , Miniaturização
12.
Proc Natl Acad Sci U S A ; 104(10): 4218-22, 2007 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-17360503

RESUMO

Folate deficiency leads to neural tube defects and other human diseases, and is a global health problem. Because plants are major folate sources for humans, we have sought to enhance plant folate levels (biofortification). Folates are synthesized from pteridine, p-aminobenzoate (PABA), and glutamate precursors. Previously, we increased pteridine production in tomato fruit up to 140-fold by overexpressing GTP cyclohydrolase I, the first enzyme of pteridine synthesis. This strategy increased folate levels 2-fold, but engineered fruit were PABA-depleted. We report here the engineering of fruit-specific overexpression of aminodeoxychorismate synthase, which catalyzes the first step of PABA synthesis. The resulting fruit contained an average of 19-fold more PABA than controls. When transgenic PABA- and pteridine-overproduction traits were combined by crossing, vine-ripened fruit accumulated up to 25-fold more folate than controls. Folate accumulation was almost as high (up to 15-fold) in fruit harvested green and ripened by ethylene-gassing, as occurs in commerce. The accumulated folates showed normal proportions of one-carbon forms, with 5-methyltetrahydrofolate the most abundant, but were less extensively polyglutamylated than controls. Folate concentrations in developing fruit did not change in controls, but increased continuously throughout ripening in transgenic fruit. Pteridine and PABA levels in transgenic fruit were >20-fold higher than in controls, but the pathway intermediates dihydropteroate and dihydrofolate did not accumulate, pointing to a flux constraint at the dihydropteroate synthesis step. The folate levels we achieved provide the complete adult daily requirement in less than one standard serving.


Assuntos
Ácido Fólico/metabolismo , Alimentos Fortificados , Lycopersicon esculentum/metabolismo , Carbono/metabolismo , Carbono-Nitrogênio Ligases/biossíntese , Carbono-Nitrogênio Ligases/genética , Cruzamentos Genéticos , Etilenos/farmacologia , Engenharia Genética/métodos , Técnicas Genéticas , Vetores Genéticos , Plantas Geneticamente Modificadas , Pteridinas/metabolismo , RNA de Plantas/metabolismo , Tetra-Hidrofolatos/farmacologia , Transaminases , Vitaminas/metabolismo
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