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1.
Medicine (Baltimore) ; 98(7): e14309, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30762730

RESUMO

Coronary heart disease (CHD), one of the leading causes of death in the world, is a complex metabolic disorder due to genetic and environmental interactions. The potential mechanisms and diagnostic biomarkers for different types of coronary heart disease remain unclear. Metabolomics is increasingly considered to be a promising technology with the potential to identify metabolomic features in an attempt to distinguish the different stages of CHD.We aimed to investigate serum metabolite profiling between CHD patients and normal coronary artery (NCA) subjects and identify metabolic biomarkers associated with CHD progression in an ethnic Hakka population in southern China.Using a novel targeted metabolomics approach, we explored the metabolic characteristics of CHD patients. Blood samples from 302 patients with CHD and 59 NCA subjects were collected that analyses using targeted liquid-chromatography coupled with tandem mass spectrometry (LC-MS).A total of 361 blood samples were determined using targeted LC-MS. Plasma concentrations for trimetlylamine oxide (TMAO), choline, creatinine, and carnitine were significantly higher in patients with CHD compared to the NCA cohort. Further, we observed that the concentration of the 4 metabolites were higher than that of the NCA group in any group of CHD, which including acute myocardial infarction (AMI), unstable angina (UA), and stable angina (SA). In addition, the diagnostic model was constructed based on the metabolites identified and the ROC curve of the NCA subjects and CHD patients were performed. For choline and creatinine, the AUCs ranged from 0.720 to 0.733. For TMAO and carnitine, the AUCs ranged from 0.568 to 0.600.In conclusion, the current study illustrates the distribution of 4 metabolites between CHD patients and NCA subjects. Metabolomics analysis may yield novel predictive biomarkers that will potentially provide value for clinical diagnosis of CHD.


Assuntos
Angina Estável/metabolismo , Metabolômica/métodos , Infarto do Miocárdio/metabolismo , Idoso , Angina Estável/sangue , Angina Estável/diagnóstico , Angina Instável/metabolismo , Biomarcadores , Carnitina/biossíntese , China , Colina/biossíntese , Cromatografia Líquida , Creatinina/sangue , Feminino , Humanos , Masculino , Metilaminas/sangue , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/diagnóstico , Estudos Prospectivos , Curva ROC , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
2.
Talanta ; 183: 258-267, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29567174

RESUMO

A highly sensitive electrochemical biosensor based on the synthetized L-Cysteine-Ag(I) coordination polymer (L-Cys-Ag(I) CP), which looks like a protein-mimicking nanowire, was constructed to detect acetylcholinesterase (AChE) activity and screen its inhibitors. This sensing strategy involves the reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce hydrogen peroxide (H2O2), thus L-Cys-Ag(I) CP possesses the electro-catalytic property to H2O2 reduction. Herein, the protein-mimicking nanowire-based platform was capable of investigating successive of H2O2 effectively by amperometric i-t (current-time) response, and was further applied for the turn-on electrochemical detection of AChE activity. The proposed sensor is highly sensitive (limit of detection is 0.0006 U/L) and is feasible for screening inhibitors of AChE. The model for AChE inhibition was further established and two traditional AChE inhibitors (donepezil and tacrine) were employed to verify the feasibility of the system. The IC50 of donepezil and tacrine were estimated to be 1.4 nM and 3.5 nM, respectively. The developed protocol provides a new and promising platform for probing AChE activity and screening its inhibitors with low cost, high sensitivity and selectivity.


Assuntos
Acetilcolinesterase/metabolismo , Técnicas Biossensoriais , Inibidores da Colinesterase/farmacologia , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Técnicas Eletroquímicas , Nanofios/química , Acetilcolina/química , Acetilcolina/metabolismo , Biocatálise , Colina/biossíntese , Colina/química , Inibidores da Colinesterase/química , Cisteína/química , Cisteína/farmacologia , Eletrodos , Polímeros/química , Polímeros/farmacologia , Prata/química , Prata/farmacologia
3.
Blood Cancer J ; 7(7): e0, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28686226

RESUMO

The activation of oncogenes can reprogram tumor cell metabolism. Here, in diffuse large B-cell lymphoma (DLBCL), serum metabolomic analysis revealed that oncogenic MYC could induce aberrant choline metabolism by transcriptionally activating the key enzyme phosphate cytidylyltransferase 1 choline-α (PCYT1A). In B-lymphoma cells, as a consequence of PCYT1A upregulation, MYC impeded lymphoma cells undergo a mitophagy-dependent necroptosis. In DLBCL patients, overexpression of PCYT1A was in parallel with an increase in tumor MYC, as well as a decrease in serum choline metabolite phosphatidylcholine levels and an International Prognostic Index, indicating intermediate-high or high risk. Both in vitro and in vivo, lipid-lowering alkaloid berberine (BBR) exhibited an anti-lymphoma activity through inhibiting MYC-driven downstream PCYT1A expression and inducing mitophagy-dependent necroptosis. Collectively, PCYT1A was upregulated by MYC, which resulted in the induction of aberrant choline metabolism and the inhibition of B-lymphoma cell necroptosis. Referred as a biomarker for DLBCL progression, PCYT1A can be targeted by BBR, providing a potential lipid-modifying strategy in treating MYC-High lymphoma.


Assuntos
Colina/biossíntese , Linfoma Difuso de Grandes Células B/metabolismo , Mitofagia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Berberina/farmacologia , Linhagem Celular Tumoral , Colina/genética , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-myc/genética
4.
Br J Pharmacol ; 173(18): 2766-79, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27423041

RESUMO

BACKGROUND AND PURPOSE: The colonic surface epithelium produces acetylcholine, released after the binding of propionate to GPCRs for this short-chain fatty acid (SCFA). This epithelial acetylcholine then induces anion secretion via stimulation of acetylcholine receptors. The key enzyme responsible for acetylcholine synthesis, choline acetyltransferase, is known to be unselective as regards the fatty acid used for esterification of choline. As the colonic epithelium is permanently exposed to high concentrations of different SCFAs produced by bacterial fermentation, we investigated whether choline esters other than acetylcholine, propionylcholine and butyrylcholine, are produced by the colonic epithelium, too, and whether these 'atypical' esters are able to stimulate the acetylcholine receptors involved in the regulation of colonic ion transport. EXPERIMENTAL APPROACH: Desorption electrospray ionization mass spectroscopy (DESI-MS), Ussing chamber and Ca(2+) -imaging experiments were performed on rat distal colon. KEY RESULTS: DESI-MS analyses revealed the production of acetylcholine, propionylcholine and butyrylcholine in the surface epithelium. Relative expression rates were 2-3% in comparison with acetylcholine. In Ussing chamber experiments, both atypical choline esters caused a concentration-dependent increase in short-circuit current, that is, stimulated anion secretion. Inhibitor experiments in the absence and presence of the submucosal plexus revealed the involvement of neuronal and epithelial acetylcholine receptors. While butyrylcholine obviously stimulated both nicotinic and muscarinic receptors, propionylcholine predominantly acted on muscarinic receptors. CONCLUSIONS AND IMPLICATIONS: These results suggest a novel pathway for communication between intestinal microbes producing SCFA and the host via modification of epithelial production of choline esters involved in the paracrine regulation of the colonic epithelium.


Assuntos
Colina/análogos & derivados , Colo/efeitos dos fármacos , Células Epiteliais/química , Animais , Colina/biossíntese , Colina/farmacologia , Colo/metabolismo , Células Epiteliais/metabolismo , Feminino , Transporte de Íons/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por Electrospray
5.
Sci Rep ; 6: 19076, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26743949

RESUMO

Many studies suggest that trimethylamine-N-oxide (TMAO), a gut-flora-dependent metabolite of choline, contributes to the risk of cardiovascular diseases, but little is known for non-alcoholic fatty liver disease (NAFLD). We examined the association of circulating TMAO, choline and betaine with the presence and severity of NAFLD in Chinese adults. We performed a hospital-based case-control study (CCS) and a cross-sectional study (CSS). In the CCS, we recruited 60 biopsy-proven NAFLD cases and 35 controls (18-60 years) and determined serum concentrations of TMAO, choline and betaine by HPLC-MS/MS. For the CSS, 1,628 community-based adults (40-75 years) completed the blood tests and ultrasonographic NAFLD evaluation. In the CCS, analyses of covariance showed adverse associations of ln-transformed serum levels of TMAO, choline and betaine/choline ratio with the scores of steatosis and total NAFLD activity (NAS) (all P-trend <0.05). The CSS revealed that a greater severity of NAFLD was independently correlated with higher TMAO but lower betaine and betaine/choline ratio (all P-trend <0.05). No significant choline-NAFLD association was observed. Our findings showed adverse associations between the circulating TMAO level and the presence and severity of NAFLD in hospital- and community-based Chinese adults, and a favorable betaine-NAFLD relationship in the community-based participants.


Assuntos
Betaína/sangue , Colina/sangue , Microbioma Gastrointestinal , Metilaminas/sangue , Hepatopatia Gordurosa não Alcoólica/patologia , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Colina/biossíntese , Cromatografia Líquida de Alta Pressão , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Índice de Gravidade de Doença , Espectrometria de Massas em Tandem , Ultrassonografia
6.
Epigenomics ; 7(7): 1173-84, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26585860

RESUMO

Children exposed to early-life adversity carry a greater risk of poor health and disease into adulthood. This increased disease risk is shadowed by changes in the epigenome. Epigenetics can change gene expression to modify disease risk; unfortunately, how epigenetics are changed by the environment is unclear. It is known that the environment modifies the microbiota, and recent data indicate that the microbiota and the epigenome interact and respond to each other. Specifically, the microbiome may alter the epigenome through the production of metabolites. Investigating the relationship between the microbiome and the epigenome may provide novel understanding of the impact of early-life environment on long-term health.


Assuntos
Epigênese Genética/efeitos dos fármacos , Genoma Humano , Microbiota/fisiologia , Probióticos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Colina/biossíntese , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Metilação de DNA , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Ácidos Graxos Voláteis/biossíntese , Ácido Fólico/biossíntese , Interação Gene-Ambiente , Histonas/genética , Histonas/imunologia , Humanos , Isotiocianatos/metabolismo , Polifenóis/biossíntese , Probióticos/metabolismo
7.
PLoS One ; 10(1): e0116470, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25606856

RESUMO

We analyzed and compared the difference in sinapine concentration in rapeseed meal between the filamentous fungus, Trametes sp 48424, and the yeast, Saccharomyces cerevisiae, in both liquid and solid-state fermentation. During liquid and solid-state fermentation by Trametes sp 48424, the sinapine concentration decreased significantly. In contrast, the liquid and solid-state fermentation process by Saccharomyces cerevisiae just slightly decreased the sinapine concentration (P ≤ 0.05). After the solid-state fermented samples were dried, the concentration of sinapine in rapeseed meal decreased significantly in Saccharomyces cerevisiae. Based on the measurement of laccase activity, we observed that laccase induced the decrease in the concentration of sinapine during fermentation with Trametes sp 48424. In order to eliminate the influence of microorganisms and the metabolites produced during fermentation, high moisture rapeseed meal and the original rapeseed meal were dried at 90 °C and 105 °C, respectively. During drying, the concentration of sinapine in high moisture rapeseed meal decreased rapidly and we obtained a high correlation coefficient between the concentration of sinapine and loss of moisture. Our results suggest that drying and enzymes, especially laccase that is produced during the solid-state fermentation process, may be the main factors that affect the concentration of sinapine in rapeseed meal.


Assuntos
Brassica rapa/química , Colina/análogos & derivados , Proteínas Fúngicas/metabolismo , Lacase/metabolismo , Brassica rapa/metabolismo , Colina/biossíntese , Fermentação , Umidade , Saccharomyces cerevisiae/enzimologia , Trametes/enzimologia
8.
Theor Appl Genet ; 124(5): 957-69, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22198204

RESUMO

We developed two mutant populations of oilseed rape (Brassica napus L.) using EMS (ethylmethanesulfonate) as a mutagen. The populations were derived from the spring type line YN01-429 and the winter type cultivar Express 617 encompassing 5,361 and 3,488 M(2) plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8× pooling strategy. Genes of the sinapine biosynthesis pathway were chosen for determining the mutation frequencies and for creating novel genetic variation for rapeseed breeding. The extraction meal of oilseed rape is a rich protein source containing about 40% protein. Its use as an animal feed or human food, however, is limited by antinutritive compounds like sinapine. The targeting-induced local lesions in genomes (TILLING) strategy was applied to identify mutations of major genes of the sinapine biosynthesis pathway. We constructed locus-specific primers for several TILLING amplicons of two sinapine synthesis genes, BnaX.SGT and BnaX.REF1, covering 80-90% of the coding sequences. Screening of both populations revealed 229 and 341 mutations within the BnaX.SGT sequences (135 missense and 13 nonsense mutations) and the BnaX.REF1 sequences (162 missense, 3 nonsense, 8 splice site mutations), respectively. These mutants provide a new resource for breeding low-sinapine oilseed rape. The frequencies of missense and nonsense mutations corresponded to the frequencies of the target codons. Mutation frequencies ranged from 1/12 to 1/22 kb for the Express 617 population and from 1/27 to 1/60 kb for the YN01-429 population. Our TILLING resource is publicly available. Due to the high mutation frequencies in combination with an 8× pooling strategy, mutants can be routinely identified in a cost-efficient manner. However, primers have to be carefully designed to amplify single sequences from the polyploid rapeseed genome.


Assuntos
Brassica napus/genética , Cruzamento/métodos , Colina/análogos & derivados , Variação Genética , Mutação/genética , Colina/biossíntese , Colina/genética , Cruzamentos Genéticos , Primers do DNA/genética , Metanossulfonato de Etila , Mutagênese/genética
9.
Neurology ; 77(10): 951-8, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21865577

RESUMO

OBJECTIVE: To determine the relationship between proton magnetic resonance spectroscopy ((1)H MRS) metabolites and ß-amyloid (Aß) load and the effects of Aß load on the association between (1)H MRS metabolites and cognitive function in cognitively normal older adults. METHODS: We studied 311 cognitively normal older adults who participated in the population-based Mayo Clinic Study of Aging from January 2009 through September 2010. Participants underwent (11)C-Pittsburgh compound B (PiB) PET, (1)H MRS from the posterior cingulate gyri, and neuropsychometric testing to assess memory, attention/executive, language, and visual-spatial domain functions within 6 months. Partial Spearman rank order correlations were adjusted for age, sex, and education. RESULTS: Higher PiB retention was associated with abnormal elevations in myoinositol (mI)/creatine (Cr) (partial r(s) = 0.17; p = 0.003) and choline (Cho)/Cr (partial r(s) = 0.13; p = 0.022) ratios. Higher Cho/Cr was associated with worse performance on Auditory Verbal Learning Test Delayed Recall (partial r(s) = -0.12; p = 0.04), Trail Making Test Part B (partial r(s) = 0.12; p = 0.04), Wechsler Adult Intelligence Scale-Revised (WAIS-R) Digit Symbol (partial r(s) = -0.18; p < 0.01), and WAIS-R Block Design (partial r(s) = -0.12; p = 0.03). Associations between (1)H MRS metabolites and cognitive function were not different among participants with high vs low PiB retention. CONCLUSION: In cognitively normal older adults, the (1)H MRS metabolite ratios mI/Cr and Cho/Cr are associated with the preclinical pathologic processes in the Alzheimer disease cascade. Higher Cho/Cr is associated with worse performance on domain-specific cognitive tests independent of Aß load, suggesting that Cho/Cr elevation may also be dependent on other preclinical dementia pathologies characterized by Cho/Cr elevation such as Lewy body or ischemic vascular disease in addition to Aß load.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Cognição/fisiologia , Espectroscopia de Ressonância Magnética , Vigilância da População , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/efeitos adversos , Colina/biossíntese , Colina/metabolismo , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/psicologia , Creatinina/metabolismo , Feminino , Humanos , Espectroscopia de Ressonância Magnética/métodos , Masculino , Testes Neuropsicológicos , Vigilância da População/métodos , Estudos Prospectivos , Estabilidade Proteica
10.
BMC Syst Biol ; 5: 12, 2011 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-21251315

RESUMO

BACKGROUND: Chromohalobacter salexigens (formerly Halomonas elongata DSM 3043) is a halophilic extremophile with a very broad salinity range and is used as a model organism to elucidate prokaryotic osmoadaptation due to its strong euryhaline phenotype. RESULTS: C. salexigens DSM 3043's metabolism was reconstructed based on genomic, biochemical and physiological information via a non-automated but iterative process. This manually-curated reconstruction accounts for 584 genes, 1386 reactions, and 1411 metabolites. By using flux balance analysis, the model was extensively validated against literature data on the C. salexigens phenotypic features, the transport and use of different substrates for growth as well as against experimental observations on the uptake and accumulation of industrially important organic osmolytes, ectoine, betaine, and its precursor choline, which play important roles in the adaptive response to osmotic stress. CONCLUSIONS: This work presents the first comprehensive genome-scale metabolic model of a halophilic bacterium. Being a useful guide for identification and filling of knowledge gaps, the reconstructed metabolic network iOA584 will accelerate the research on halophilic bacteria towards application of systems biology approaches and design of metabolic engineering strategies.


Assuntos
Chromohalobacter/genética , Chromohalobacter/metabolismo , Genômica/métodos , Redes e Vias Metabólicas/genética , Adaptação Fisiológica , Diamino Aminoácidos/biossíntese , Betaína/metabolismo , Colina/biossíntese , Chromohalobacter/fisiologia , Genoma Bacteriano/genética , Modelos Biológicos , Fenótipo
11.
J Biol Chem ; 286(2): 1649-58, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21059658

RESUMO

When dietary choline is restricted, most men and postmenopausal women develop multiorgan dysfunction marked by hepatic steatosis (choline deficiency syndrome (CDS)). However, a significant subset of premenopausal women is protected from CDS. Because hepatic PEMT (phosphatidylethanolamine N-methyltransferase) catalyzes de novo biosynthesis of choline and this gene is under estrogenic control, we hypothesized that there are SNPs in PEMT that disrupt the hormonal regulation of PEMT and thereby put women at risk for CDS. In this study, we performed transcript-specific gene expression analysis, which revealed that estrogen regulates PEMT in an isoform-specific fashion. Locus-wide SNP analysis identified a risk-associated haplotype that was selectively associated with loss of hormonal activation. Chromatin immunoprecipitation, analyzed by locus-wide microarray studies, comprehensively identified regions of estrogen receptor binding in PEMT. The polymorphism (rs12325817) most highly linked with the development of CDS (p < 0.00006) was located within 1 kb of the critical estrogen response element. The risk allele failed to bind either the estrogen receptor or the pioneer factor FOXA1. These data demonstrate that allele-specific ablation of estrogen receptor-DNA interaction in the PEMT locus prevents hormone-inducible PEMT expression, conferring risk of CDS in women.


Assuntos
Deficiência de Colina/metabolismo , Estrogênios/metabolismo , Hepatopatias/metabolismo , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Células Cultivadas , Colina/biossíntese , Deficiência de Colina/epidemiologia , Deficiência de Colina/fisiopatologia , Feminino , Predisposição Genética para Doença/epidemiologia , Haplótipos , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Hepatopatias/epidemiologia , Hepatopatias/fisiopatologia , Masculino , Insuficiência de Múltiplos Órgãos/epidemiologia , Insuficiência de Múltiplos Órgãos/metabolismo , Insuficiência de Múltiplos Órgãos/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Pré-Menopausa/fisiologia , Receptores Estrogênicos/metabolismo , Fatores de Risco , Transcrição Genética/fisiologia
12.
Mol Plant Microbe Interact ; 23(8): 1097-104, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20615119

RESUMO

Choline (Cho) is an essential nutrient for humans as well as the precursor of glycine betaine (GlyBet), an important compatible solute in eukaryotes that protects cells from osmotic stress caused by dehydrating conditions. The key enzyme for plant Cho synthesis is phosphoethanolamine N-methyltransferase (PEAMT), which catalyzes all three methylation steps, including the rate-limiting N-methylation of phosphoethanolamine. Herein, we report that the beneficial soil bacterium Bacillus subtilis (strain GB03) enhances Arabidopsis Cho and GlyBet synthesis associated with enhanced plant tolerance to osmotic stress. When stressed with 100 mM exogenous mannitol, GB03-exposed plants exhibit increased transcript level of PEAMT compared with stressed plants without bacterial exposure. Endogenous Cho and GlyBet metabolite pools were elevated by more than two- and fivefold, respectively, by GB03 treatment, consistent with increased stress tolerance. Moreover, in the xipotl mutant line with reduced Cho production, a loss of GB03-induced drought tolerance is observed. Osmotic-stressed plants with or without GB03 exposure show similar levels of abscsisic acid (ABA) accumulation in both shoots and roots, suggesting that GB03-induced osmoprotection is ABA independent. GB03 treatment also improves drought tolerance in soil-grown plants as characterized by phenotypic comparisons, supported by an elevated accumulation of osmoprotectants. These results provide a biological strategy to enhance Cho biosynthesis in plants and, in turn, increase plant tolerance to osmotic stress by elevating osmoprotectant accumulation.


Assuntos
Arabidopsis/microbiologia , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Colina/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/fisiologia , Betaína/metabolismo , Colina/biossíntese , Primers do DNA , Etanolamina/metabolismo , Etanolaminofosfotransferase/genética , Etanolaminofosfotransferase/metabolismo , Humanos , Cinética , Pressão Osmótica , Folhas de Planta/fisiologia , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Genética
13.
J Biol Chem ; 285(29): 22403-13, 2010 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-20452975

RESUMO

Phosphatidylcholine (PC) is synthesized from choline via the CDP-choline pathway. Liver cells can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). The current study investigates whether or not hepatic PC biosynthesis is linked to diet-induced obesity. Pemt(+/+) mice fed a high fat diet for 10 weeks increased in body mass by 60% and displayed insulin resistance, whereas Pemt(-/-) mice did not. Compared with Pemt(+/+) mice, Pemt(-/-) mice had increased energy expenditure and maintained normal peripheral insulin sensitivity; however, they developed hepatomegaly and steatosis. In contrast, mice with impaired biosynthesis of PC via the CDP-choline pathway in liver became obese when fed a high fat diet. We, therefore, hypothesized that insufficient choline, rather than decreased hepatic phosphatidylcholine, was responsible for the lack of weight gain in Pemt(-/-) mice despite the presence of 1.3 g of choline/kg high fat diet. Supplementation with an additional 2.7 g of choline (but not betaine)/kg of diet normalized energy metabolism, weight gain, and insulin resistance in high fat diet-fed Pemt(-/-) mice. Furthermore, Pemt(+/+) mice that were fed a choline-deficient diet had increased oxygen consumption, had improved glucose tolerance, and gained less weight. Thus, de novo synthesis of choline via PEMT has a previously unappreciated role in regulating whole body energy metabolism.


Assuntos
Colina/biossíntese , Dieta , Obesidade/enzimologia , Obesidade/prevenção & controle , Fosfatidiletanolamina N-Metiltransferase/deficiência , Animais , Betaína/administração & dosagem , Betaína/farmacologia , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Suplementos Nutricionais , Metabolismo Energético/efeitos dos fármacos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/complicações , Fígado Gorduroso/enzimologia , Fígado Gorduroso/patologia , Comportamento Alimentar/efeitos dos fármacos , Resistência à Insulina , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Obesidade/induzido quimicamente , Obesidade/complicações , Fenótipo , Fosfatidilcolinas/biossíntese , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Ganho de Peso/efeitos dos fármacos
14.
Planta ; 227(2): 375-85, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17882453

RESUMO

This study describes the molecular characterization of the genes BnSCT1 and BnSCT2 from oilseed rape (Brassica napus) encoding the enzyme 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT; EC 2.3.1.91). SCT catalyzes the 1-O-beta-acetal ester-dependent biosynthesis of sinapoylcholine (sinapine), the most abundant phenolic compound in seeds of B. napus. GUS fusion experiments indicated that seed specificity of BnSCT1 expression is caused by an inducible promoter confining transcription to embryo tissues and the aleurone layer. A dsRNAi construct designed to silence seed-specifically the BnSCT1 gene was effective in reducing the sinapine content of Arabidopsis seeds thus defining SCT genes as targets for molecular breeding of low sinapine cultivars of B. napus. Sequence analyses revealed that in the allotetraploid genome of B. napus the gene BnSCT1 represents the C genome homologue from the B. oleracea progenitor whereas BnSCT2 was derived from the Brassica A genome of B. rapa. The BnSCT1 and BnSCT2 loci showed colinearity with the homologous Arabidopsis SNG2 gene locus although the genomic microstructure revealed the deletion of a cluster of three genes and several coding regions in the B. napus genome.


Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Brassica napus/enzimologia , Brassica napus/genética , Colina/análogos & derivados , Aciltransferases/química , Arabidopsis/genética , Arabidopsis/metabolismo , Colina/biossíntese , Regulação da Expressão Gênica de Plantas , Mutação , Filogenia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Interferência de RNA , Tabaco/citologia
15.
FASEB J ; 21(14): 4038-46, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17627030

RESUMO

The chemokine receptor CCR5 plays an important role as an entry gate for the human immunodeficiency virus-1 (HIV-1) and for viral postentry events. Among signal transducers used by chemoattractant receptors, the phosphatidylcholine-specific phospholipase D (PLD) produces large amounts of second messengers in most cell types. However, the relevance of PLD isoforms to CCR5 signaling and HIV-1 infection process remains unexplored. We show here that CCR5 activation by MIP-1beta in HeLa-MAGI cells triggered a rapid and substantial PLD activity, as assessed by mass choline production. This activity required the activation of ERK1/2-MAP kinases and involved both PLD1 and PLD2. MIP-1beta also promoted the activation of an HIV-1 long terminal repeat (LTR) by the transactivator Tat in HeLa P4.2 cells through a process involving ERK1/2. Expression of wild-type and catalytically inactive PLDs dramatically boosted and inhibited the LTR activation, respectively, without altering Tat expression. Wild-type and inactive PLDs also respectively potentiated and inhibited HIV-1(BAL) replication in MAGI cells. Finally, in monocytic THP-1 cells, antisense oligonucleotides to both PLDs dramatically inhibited the HIV-1 replication. Thus, PLD is activated downstream of ERK1/2 upon CCR5 activation and plays a major role in promoting HIV-1 LTR transactivation and virus replication, which may open novel perspectives to anti-HIV-1 strategies.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Repetição Terminal Longa de HIV/fisiologia , HIV-1/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Fosfolipase D/fisiologia , Receptores CCR5/fisiologia , Colina/biossíntese , HIV-1/enzimologia , HIV-1/fisiologia , Células HeLa , Humanos , Fosfolipase D/metabolismo , Receptores CCR5/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional/genética , Replicação Viral/fisiologia
16.
J Biotechnol ; 130(2): 133-42, 2007 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-17475356

RESUMO

A comprehensive metabolomic profiling of Silybum marianum (L.) Gaernt cell cultures elicited with yeast extract or methyl jasmonate for the production of silymarin was carried out using one- and two-dimensional nuclear magnetic resonance spectroscopy. With these techniques we were able to detect both temporal quantitative variations in the metabolite pool in yeast extract-elicited cultures and qualitative differences in cultures treated with the two types of elicitors. Yeast extract and methyl jasmonate caused a metabolic reprogramming that affected amino acid and carbohydrate metabolism; upon elicitation sucrose decreased and glucose levels increased, these changes being dependent on "de novo" protein synthesis. Also dependent on protein synthesis were the increase seen in alanine and glutamine in elicited cultures. Yeast extract differentially acted on threonine and valine metabolism and promoted accumulation of choline and alpha-linolenic acid in cells thus suggesting its action on membranes and the involvement of the octadecanoid pathway in the induction of silymarin in S. marianum cultures. Phenylpropanoid metabolism was altered by elicitation but, depending on elicitor, different phenylpropanoid profile was produced. The results obtained in this study will permit in the future to identify candidate components of the signalling pathway involved in the stimulation of the constitutive pathway of silymarin.


Assuntos
Acetatos/farmacologia , Ciclopentanos/farmacologia , Cardo-Mariano/citologia , Cardo-Mariano/metabolismo , Ressonância Magnética Nuclear Biomolecular , Reguladores de Crescimento de Planta/farmacologia , Alanina/biossíntese , Aminoácidos/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Técnicas de Cultura de Células , Células Cultivadas , Colina/biossíntese , Cicloeximida/farmacologia , Glucose/metabolismo , Ácido Glutâmico/biossíntese , Oxilipinas , Inibidores da Síntese de Proteínas/farmacologia , Silimarina/metabolismo , Sacarose/metabolismo , Leveduras/química , Ácido alfa-Linoleico/biossíntese
17.
Annu Rev Nutr ; 26: 229-50, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16848706

RESUMO

Choline is an essential nutrient needed for the structural integrity and signaling functions of cell membranes; for normal cholinergic neurotransmission; for normal muscle function; for lipid transport from liver; and it is the major source of methyl groups in the diet. Choline is critical during fetal development, when it influences stem cell proliferation and apoptosis, thereby altering brain and spinal cord structure and function and influencing risk for neural tube defects and lifelong memory function. Choline is derived not only from the diet, but from de novo synthesis as well. Though many foods contain choline, there is at least a twofold variation in dietary intake in humans. When deprived of dietary choline, most men and postmenopausal women developed signs of organ dysfunction (fatty liver or muscle damage), while less than half of premenopausal women developed such signs. Aside from gender differences, there is significant variation in the dietary requirement for choline that can be explained by very common genetic polymorphisms.


Assuntos
Deficiência de Colina/fisiopatologia , Colina/administração & dosagem , Colina/fisiologia , Desenvolvimento Fetal/efeitos dos fármacos , Necessidades Nutricionais , Polimorfismo Genético , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Colina/biossíntese , Colina/genética , Deficiência de Colina/genética , Feminino , Humanos , Masculino , Gravidez , Fatores Sexuais
18.
Acta Pharmacol Sin ; 25(12): 1652-8, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15569411

RESUMO

AIM: To investigate the effect of vasoactive intestinal peptide (VIP) on pulmonary surfactants (PS) phospholipid synthesis in cultured lung explants. METHODS: Lung explants were cultured with serum-free medium, [methyl-3H]choline incorporation, total phospholipid, phosphatidylcholine, activity of choline-phosphate cytidylyltransferase (CCT) and CCTalpha mRNA level in lung explants were determined. RESULTS: (1) VIP (10(-10)-10(-7) mol/L) for 16 h promoted [methyl-3H]choline incorporation in dose dependence and VIP (10(-8) mol/L) for 2 h-16 h promoted [methyl-3H]choline incorporation in time dependence. (2) VIP (10(-8) mol/L) enhanced the contents of total phospholipids and phosphatidylcholine in lung explants. (3) VIP (10(-10)-10(-7) mol/L) elevated microsomal CCT activity of lung explants in dose dependence. (4) VIP (10(-8) mol/L) increased expression of CCTalpha mRNA in lung explants and alveolar type II cells (ATII). (5) [D-P-Cl-Phe(6)-Leu(17)]-VIP (10(-6) mol/L), a VIP receptors antagonist, abolished the increase of [3H]choline incorporation, microsomal CCT activity and CCTalpha mRNA level induced by VIP (10(-8) mol/L) in lung explants. CONCLUSION: VIP could enhance synthesis of phosphatidylcholine, the major component of pulmonary surfactants by enhancing microsomal CCT activity and CCTalpha mRNA level via VIP receptor-mediated pathway.


Assuntos
Colina-Fosfato Citidililtransferase/biossíntese , Pulmão/enzimologia , Surfactantes Pulmonares/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Células Cultivadas , Colina/biossíntese , Colina-Fosfato Citidililtransferase/genética , Feminino , Isoenzimas/biossíntese , Isoenzimas/genética , Pulmão/citologia , Masculino , Microssomos/enzimologia , Fosfatidilcolinas/biossíntese , Fosfolipídeos/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores de Peptídeo Intestinal Vasoativo/antagonistas & inibidores
19.
Lipids ; 38(1): 45-52, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12669819

RESUMO

In pericytes from bovine retina, the enzyme glycerophosphocholine phosphodiesterase, catalyzing the hydrolysis of sn-glycero-3-phosphocholine to glycero-3-phosphate and choline, has been characterized with respect to pH optimum, metal ion dependence, Km, inhibitors, and subcellular localization. In these cells, the natural substrate sn-glycero-3-phosphocholine was present at relatively high concentration (6.4 +/- 1.2 nmol/mg protein), and the EDTA-sensitive phosphodiesterase activity was also found to be markedly high (9.80 +/- 1.5 nmol/min/mg protein) compared to that estimated in liver and brain (1-3 nmol/min/mg protein) or in renal epithelial cell culture (0.27 nmol/min/mg protein). The reaction conditions were in general agreement with those found earlier in brain and other tissues. The majority of the enzyme specific activity was located in the plasma membrane, whereas a minor part was present in the microsomal fraction. The physiological significance of the high catabolic phosphodiesterase activity in these cells may be related to the transfer, followed by deacylation, of lysophosphatidylcholine from the bloodstream to nervous tissue. In addition, capillary pericytes in culture were able to incorporate 3H-choline rapidly into choline-containing soluble phosphorylated intermediates and into phosphatidylcholine. To find a positive and negative effector on phosphatidylcholine formation, adenosine, an important intercellular mediator in the retina in response to alterations in oxygen delivery, and endothelin-1, a potent paracrine mediator present at the blood-brain and blood-retina barrier, were tested. The cells cultured for 1 or 24 h in a medium containing adenosine at concentrations of 10(-6) and 10(-4) M showed significant reduction in 3H-choline incorporation compared to control cultures, whereas endothelin-1, at a concentration of 10 and 100 nM, caused stimulation of phosphatidylcholine biosynthesis. These findings provide evidence that both agonists may modulate phosphatidylcholine metabolism in pericytes.


Assuntos
Adenosina/farmacologia , Endotelina-1/farmacologia , Pericitos/enzimologia , Fosfatidilcolinas/biossíntese , Diester Fosfórico Hidrolases/metabolismo , Vasos Retinianos/citologia , Animais , Barreira Hematorretiniana , Capilares/citologia , Cátions/metabolismo , Bovinos , Células Cultivadas , Colina/biossíntese , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Fosfolipídeos/metabolismo
20.
Plant Cell ; 14(9): 2031-43, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12215503

RESUMO

S-Adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT; EC 2.1.1.103) catalyzes the key step in choline (Cho) biosynthesis, the N-methylation of phosphoethanolamine. Cho is a vital precursor of the membrane phospholipid phosphatidylcholine, which accounts for 40 to 60% of lipids in nonplastid plant membranes. Certain plants use Cho to produce the osmoprotectant glycine betaine, which confers resistance to salinity, drought, and other stresses. An Arabidopsis mutant, t365, in which the PEAMT gene is silenced, was identified using a new sense/antisense RNA expression system. t365 mutant plants displayed multiple morphological phenotypes, including pale-green leaves, early senescence, and temperature-sensitive male sterility. Moreover, t365 mutant plants produced much less Cho and were hypersensitive to salinity. These results demonstrate that Cho biosynthesis not only plays an important role in plant growth and development but also contributes to tolerance to environmental stresses. The temperature-sensitive male sterility caused by PEAMT silencing may have a potential application in agriculture for engineering temperature-sensitive male sterility in important crop plants.


Assuntos
Arabidopsis/fisiologia , Inativação Gênica/fisiologia , Metiltransferases/metabolismo , Pólen/crescimento & desenvolvimento , Adaptação Fisiológica , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Colina/biossíntese , Clonagem Molecular , Fertilidade/genética , Fertilidade/fisiologia , Metiltransferases/efeitos dos fármacos , Metiltransferases/genética , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Pólen/genética , Cloreto de Sódio/farmacologia , Temperatura
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