RESUMO
Cutibacterium acnes (formerly Propionibacterium acnes) is one of the major bacterial species responsible for acne vulgaris. Numerous bioactive compounds from Momordica charantia Linn. var. abbreviata Ser. have been isolated and examined for many years. In this study, we evaluated the suppressive effect of two cucurbitane-type triterpenoids, 5ß,19-epoxycucurbita-6,23-dien-3ß,19,25-triol (Kuguacin R; KR) and 3ß,7ß,25-trihydroxycucurbita-5,23-dien-19-al (TCD) on live C. acnes-stimulated in vitro and in vivo inflammatory responses. Using human THP-1 monocytes, KR or TCD suppressed C. acnes-induced production of interleukin (IL)-1ß, IL-6 and IL-8 at least above 56% or 45%, as well as gene expression of these three pro-inflammatory cytokines. However, a significantly strong inhibitory effect on production and expression of tumor necrosis factor (TNF)-α was not observed. Both cucurbitanes inhibited C. acnes-induced activation of the myeloid differentiation primary response 88 (MyD88) (up to 62%) and mitogen-activated protein kinases (MAPK) (at least 36%). Furthermore, TCD suppressed the expression of pro-caspase-1 and cleaved caspase-1 (p10). In a separate study, KR or TCD decreased C. acnes-stimulated mouse ear edema by ear thickness (20% or 14%), and reduced IL-1ß-expressing leukocytes and neutrophils in mouse ears. We demonstrated that KR and TCD are potential anti-inflammatory agents for modulating C. acnes-induced inflammation in vitro and in vivo.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Cucurbitacinas/química , Cucurbitacinas/farmacologia , Inflamação/tratamento farmacológico , Momordica charantia/química , Triterpenos/química , Triterpenos/farmacologia , Acne Vulgar/tratamento farmacológico , Acne Vulgar/imunologia , Acne Vulgar/microbiologia , Animais , Citocinas/biossíntese , Citocinas/genética , Modelos Animais de Doenças , Glicosídeos/química , Glicosídeos/farmacologia , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Propionibacteriaceae/patogenicidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células THP-1RESUMO
Cutibacterium acnes (formerly Propionibacterium acnes) is a key pathogen involved in the development and progression of acne inflammation. The numerous bioactive properties of wild bitter melon (WBM) leaf extract and their medicinal applications have been recognized for many years. In this study, we examined the suppressive effect of a methanolic extract (ME) of WBM leaf and fractionated components thereof on live C. acnes-induced in vitro and in vivo inflammation. Following methanol extraction of WBM leaves, we confirmed anti-inflammatory properties of ME in C. acnes-treated human THP-1 monocyte and mouse ear edema models. Using a bioassay-monitored isolation approach and a combination of liquid-liquid extraction and column chromatography, the ME was then separated into n-hexane, ethyl acetate, n-butanol and water-soluble fractions. The hexane fraction exerted the most potent anti-inflammatory effect, suppressing C. acnes-induced interleukin-8 (IL-8) production by 36%. The ethanol-soluble fraction (ESF), which was separated from the n-hexane fraction, significantly inhibited C. acnes-induced activation of mitogen-activated protein kinase (MAPK)-mediated cellular IL-8 production. Similarly, the ESF protected against C. acnes-stimulated mouse ear swelling, as measured by ear thickness (20%) and biopsy weight (23%). Twenty-four compounds in the ESF were identified using gas chromatograph-mass spectrum (GC/MS) analysis. Using co-cultures of C. acnes and THP-1 cells, ß-ionone, a compound of the ESF, reduced the production of IL-1ß and IL-8 up to 40% and 18%, respectively. ß-ionone also reduced epidermal microabscess, neutrophilic infiltration and IL-1ß expression in mouse ear. We also found evidence of the presence of anti-inflammatory substances in an unfractionated phenolic extract of WBM leaf, and demonstrated that the ESF is a potential anti-inflammatory agent for modulating in vitro and in vivo C. acnes-induced inflammatory responses.
Assuntos
Anti-Inflamatórios/química , Momordica charantia/química , Extratos Vegetais/química , Propionibacteriaceae/patogenicidade , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Edema/tratamento farmacológico , Edema/microbiologia , Edema/patologia , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Masculino , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Momordica charantia/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/microbiologia , Extratos Vegetais/análise , Folhas de Planta/química , Folhas de Planta/metabolismoRESUMO
Acne vulgaris (acne) is a common inflammatory skin disorder, and Propionibacterium acnes plays a major role in the development and progression of acne inflammation. Herbs possessing antimicrobial and anti-inflammatory activity have been applied as a medical option for centuries. In this study, we examined the suppressive effect of ethanolic oregano (Origanum vulgare) extract on live P. acnes-induced in vivo and in vitro inflammation. Following ethanol extraction of oregano leaves, four compounds with strong antioxidant activity, including rosmarinic acid, quercetin, apigenin, and carvacrol, were identified by high-performance liquid chromatography. Using the mouse ear edema model, we demonstrated that ethanol oregano extracts (EOE) significantly suppressed P. acnes-induced skin inflammation, as measured by ear thickness (32%) and biopsy weight (37%). In a separate study, using the co-culture of P. acnes and human THP-1 monocytes, EOE reduced the production of interleukin (IL)-8, IL-1ß and tumor necrosis factor (TNF)-α up to 40%, 37%, and 18%, respectively, as well as the expression of these three pro-inflammatory mediators at the transcriptional level. Furthermore, EOE inhibited the translocation of nuclear factor-kappa B (NF-κB) into the nucleus possibly by inactivating toll-like receptor-2 (TLR2). The suppressive effect of EOE on live P. acnes-induced inflammatory responses could be due, in part, to the anti-inflammatory and antioxidant properties, but not the anti-microbial effect of EOE.
Assuntos
Orelha/patologia , Edema/tratamento farmacológico , Etanol/química , Inflamação/tratamento farmacológico , Monócitos/microbiologia , Origanum/química , Extratos Vegetais/uso terapêutico , Propionibacterium acnes/efeitos dos fármacos , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Citocinas/biossíntese , Citocinas/genética , Modelos Animais de Doenças , Edema/microbiologia , Edema/patologia , Humanos , Inflamação/microbiologia , Inflamação/patologia , Masculino , Camundongos Endogâmicos ICR , Monócitos/efeitos dos fármacos , Monócitos/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Fenóis/análise , Extratos Vegetais/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor 2 Toll-Like/metabolismoRESUMO
The excess influx of free fatty acids (FFAs) into nonadipose tissues, such as those of liver and kidney, induces lipotoxicity leading to hepatic steatosis and renal dysfunction. The aim of this study was to investigate the protective effects of methanolic flower extracts of Osmanthus fragrans (OF) and Chrysanthemum morifolium (CM) against FFA-induced lipotoxicity in hepatocytes (human HepG2 cells) and renal glomerular mesangial cells (mouse SV40-Mes13 cells). The results showed that OF and CM significantly suppressed FFA-induced intracellular triacylglycerol accumulation via partially inhibiting the gene expression of sterol regulatory element-binding protein-1c (SREBP-1c) and glycerol-3-phosphate acyltransferase (GPAT) in HepG2 cells. Both extracts inhibited reactive oxygen species (ROS) generation by FFA-stimulated HepG2 cells. OF and CM also suppressed the mRNA expression of interleukin- (IL-) 1ß, IL-6, IL-8, tumor necrosis factor- (TNF-) α, and transforming growth factor- (TGF-) ß by HepG2 cells treated with conditioned medium derived from lipopolysaccharide-treated THP-1 monocytes. Furthermore, OF and CM effectively inhibited oleate-induced cellular lipid accumulation, TGF-ß secretion, and overexpression of fibronectin in mesangial cells. In conclusion, OF and CM possess hepatoprotective activity by inhibiting hepatic fat load and inflammation and renal protection by preventing FFA-induced mesangial extracellular matrix formation.
Assuntos
Chrysanthemum , Hepatócitos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Células Mesangiais/efeitos dos fármacos , Oleaceae , Extratos Vegetais/farmacologia , Animais , Ácidos Graxos não Esterificados/farmacologia , Flores , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Células Mesangiais/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta/análise , Triglicerídeos/metabolismoRESUMO
Propionibacterium acnes is a key pathogen involved in acne inflammation. Wild bitter melon (WBM, Momordica charantia L. var. abbreviate Seringe) is consumed as both a vegetable and as folk medicine in Taiwan. We examined the inhibitory activity of the total phenolic extract (TPE) of WBM leaf on P. acnes-induced inflammatory responses in vivo and in vitro. Our data showed that TPE significantly attenuated P. acnes-induced ear swelling in mice along with microabscess. Flow cytometry analysis revealed that TPE treatment significantly decreased the migration of neutrophils and interleukin (IL)-1ß(+) populations in vivo. In P. acnes-stimulated human monocytic THP-1 cells, TPE suppressed the mRNA levels and production of IL-8, IL-1ß, and tumor necrosis factor (TNF)-αin vitro. In addition, TPE suppressed P. acnes-induced matrix metalloproteinase-9 levels. TPE blocked nuclear factor-κB (NF-κB) activation and inactivated mitogen-activated protein kinases (MAPK); these actions may partially account for its inhibitory effect on cytokine production. The quantitative HPLC analysis revealed gallic, chlorogenic, caffeic, ferulic, and cinnamic acids, myricetin, quercetin, luteolin, apigenin, and thymol in TPE. All these phenolics significantly suppressed P. acnes-induced IL-8 production in vitro. Our results suggest that WBM leaf extract effectively inhibits P. acnes-induced inflammatory responses and may be useful to relieve the inflammation of acne.
Assuntos
Acne Vulgar/tratamento farmacológico , Acne Vulgar/imunologia , Momordica charantia/química , Extratos Vegetais/administração & dosagem , Propionibacterium acnes/fisiologia , Acne Vulgar/genética , Acne Vulgar/microbiologia , Animais , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Taiwan , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Propionibacterium acnes is a key pathogen involved in the progression of acne inflammation. The development of a new agent possessing antimicrobial and anti-inflammatory activity against P. acnes is therefore of interest. In this study, we investigated the inhibitory effect of rosemary (Rosmarinus officinalis) extract on P. acnes-induced inflammation in vitro and in vivo. The results showed that ethanolic rosemary extract (ERE) significantly suppressed the secretion and mRNA expression of proinflammatory cytokines, including interleukin (IL)-8, IL-1ß, and tumor necrosis factor-α in P. acnes-stimulated monocytic THP-1 cells. In an in vivo mouse model, concomitant intradermal injection of ERE attenuated the P. acnes-induced ear swelling and granulomatous inflammation. Since ERE suppressed the P. acnes-induced nuclear factor kappa-B (NF-κB) activation and mRNA expression of Toll-like receptor (TLR) 2, the suppressive effect of ERE might be due, at least partially, to diminished NF-κB activation and TLR2-mediated signaling pathways. Furthermore, three major constituents of ERE, carnosol, carnosic acid, and rosmarinic acid, exerted different immumodulatory activities in vitro. In brief, rosmarinic acid significantly suppressed IL-8 production, while the other two compounds inhibited IL-1ß production. Further study is needed to explore the role of bioactive compounds of rosemary in mitigation of P. acnes-induced inflammation.
Assuntos
Acne Vulgar/patologia , Anti-Inflamatórios/uso terapêutico , Mediadores da Inflamação/metabolismo , Inflamação/tratamento farmacológico , Extratos Vegetais/farmacologia , Propionibacterium acnes , Rosmarinus/química , Abietanos/farmacologia , Abietanos/uso terapêutico , Acne Vulgar/tratamento farmacológico , Acne Vulgar/metabolismo , Acne Vulgar/microbiologia , Animais , Anti-Inflamatórios/farmacologia , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Citocinas/genética , Citocinas/metabolismo , Depsídeos/farmacologia , Depsídeos/uso terapêutico , Humanos , Inflamação/genética , Inflamação/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fitoterapia , Extratos Vegetais/uso terapêutico , RNA Mensageiro/metabolismo , Transdução de Sinais , Ácido RosmarínicoRESUMO
Green tea is one of the most-consumed beverages due to its taste and antioxidative polyphenols. However, the protective effects of green tea and its constituent, gallic acid (GA), against kainic acid (KA)-induced seizure have not been studied. We investigated the effect of fresh green tea leaf (GTL) and GA on KA-induced neuronal injury in vivo and in vitro. The results showed that GTL and GA reduced the maximal seizure classes, predominant behavioral seizure patterns, and lipid peroxidation in male FVB mice with status epilepticus (SE). GTL extract and GA provided effective protection against KA-stressed PC12 cells in a dose-dependent manner. In the protective mechanism study, GTL and GA decreased Ca(2+) release, ROS, and lipid peroxidation from KA-stressed PC12 cells. Western blot results revealed that mitogen-activated protein kinases (MAPKs), RhoA, and COX-2 expression were increased in PC12 cells under KA stress, and expression of COX-2 and p38 MAPK, but not RhoA, was significantly reduced by GTL and GA. Furthermore, GTL and GA were able to reduce PGE(2) production from KA-stressed PC12 cells. Taken together, the results showed that GTL and GA provided neuroprotective effects against excitotoxins and may have a clinical application in epilepsy.
Assuntos
Ácido Gálico/farmacologia , Ácido Caínico , Estresse Oxidativo/efeitos dos fármacos , Estado Epiléptico/metabolismo , Chá/química , Animais , Antioxidantes , Cálcio/metabolismo , Ácido Caínico/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Fármacos Neuroprotetores , Células PC12 , Extratos Vegetais/farmacologia , Ratos , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológicoRESUMO
OBJECTIVES: Grape-seed procyanidins (GSPs) can inhibit cell proliferation and invasiveness in various human cancers. However, the effect of GSP on pancreatic carcinoma cells has not been investigated. METHODS: Pancreatic carcinoma cell lines MIA PaCa-2 and BxPC-3 treated with GSP were assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for increased apoptosis by annexin V labeling, for their adhesion and invasion potential by evaluating their ability to penetrate through a matrix gel-coated Boyden chamber, and for changes in the levels of proteins involved in cellular events by immunoblotting. RESULTS: Grape-seed procyanidin inhibited MIA PaCa-2 and BxPC-3 proliferation in a dose-dependent manner and induced G1-phase arrest of the cell cycle in BxPC-3 or mitochondria-mediated apoptosis in MIA PaCa-2. Grape-seed procyanidin also inhibited the adhesion and invasion potential of both cell lines in a dose-dependent manner, which are associated with the suppression of metalloproteases matrix metalloproteinase 9 or 2 (MMP-9 or -2) expression. CONCLUSIONS: Grape-seed procyanidin inhibited the proliferation of pancreatic carcinoma cells by cell cycle blockage or apoptotic induction. The invasiveness was also suppressed by GSP through down-regulation of MMP-2 or MMP-9 in pancreatic carcinoma cells. Grape-seed procyanidin is a potential chemotherapeutic or preventive agent for pancreatic carcinoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Extrato de Sementes de Uva/farmacologia , Neoplasias Pancreáticas/patologia , Proantocianidinas/farmacologia , Idoso , Apoptose/efeitos dos fármacos , Western Blotting , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Invasividade NeoplásicaRESUMO
Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers derived from linoleic acid (LA: delta9, 12-18:2), has been shown to exhibit various biological functions based on studies using cell culture and animal models. It was postulated that the beneficial effects of CLA were exerted through suppression of production of arachidonic acid (AA; delta5,8,11,14-20:4) and consequently, production of pro-inflammatory eicosanoids. In this study, we used the baker's yeast, Saccharomyces cerevisiae, transformed with fungal delta5-desaturase gene as a model, to study whether CLA affects the activity of delta5-desaturase, a rate-limiting step which converts dihomo-gamma-linolenic acid (DGLA; delta8,11, 14-20:3) to AA. The activity of delta5-desaturase was examined in the transformed yeast incubated in a medium supplemented with DGLA and one of four different CLA isomers (c9, t11-, t10, c12-, c9, c11- and t9, t11). Results show that all four isomers were taken up readily by the yeast, and all of them suppressed the conversion of DGLA to AA. The degree of suppression, which varied significantly among four isomers was modulated by the level of CLA isomers added in the medium. Since portions of these CLA isomers could be converted to form delta5-CLA metabolites (delta5, c9, t11-, delta5, t10, c12-, delta5, c9, c11- and delta5, t9, t11-18:3), it is suggested that CLA suppressed the delta5-desaturation of DGLA to AA through substrate competition between DGLA and CLA isomers.
Assuntos
Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Linoleicos Conjugados/farmacologia , Saccharomyces cerevisiae/metabolismo , Ácido Araquidônico/metabolismo , Cromatografia Gasosa , Dessaturase de Ácido Graxo Delta-5 , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Técnicas Genéticas , Metabolismo dos Lipídeos , Modelos Biológicos , Plasmídeos/metabolismo , Saccharomyces cerevisiae/genética , Fatores de TempoRESUMO
Dietary supplementation of a high-gamma-linolenic acid canola oil (HGCO) containing approximately 36% (w/w) of gamma-linolenic acid (GLA, 18:3n-6) from the seeds of a genetically transformed canola strain, was assessed for its long-term biological effects. Growing Sprague-Dawley rats (n = 30) were fed a purified AIN93G diet containing 5, 10, or 15% (w/w) of HGCO as the fat source. For comparison, a separate group of rats (n = 10) was given the diet containing 15% (w/w) of borage oil (BO), which contained 22% (w/w) of GLA. After 12 weeks of feeding, the growth, relative organ weights, hematology, and serum biochemistry were found to be similar among rats fed the 5, 10, and 15% HGCO diets. The GLA levels in plasma and liver phospholipids (PL) were also similar. However, the levels of GLA in peripheral tissues (muscle PL and adipose triacylglycerols) were significantly higher in rats fed the 10 and 15% HGCO diets than those fed the 5% HGCO diet. When the above biologic parameters were compared between the 15% HGCO and 15% BO dietary groups, there were no significant differences except for lower final body weights and higher tissue levels of GLA, dihomo-gamma-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) in the 15% HGCO dietary group as compared with the 15% BO dietary group. This is due to a higher GLA content and possibly a more favorable stereospecific distribution of GLA in HGCO. Overall, long-term (12-week) feeding with diets containing up to 15% HGCO resulted in no adverse effects on growth, organ weight, hematology and serum biochemistry as compared to the diet containing 15% BO, suggesting that HGCO may be a safe alternative source of GLA.
Assuntos
Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos Monoinsaturados/administração & dosagem , Ácidos Graxos Ômega-6/metabolismo , Óleos de Plantas/administração & dosagem , Aumento de Peso , Ácido gama-Linolênico/administração & dosagem , Tecido Adiposo , Animais , Ácidos Graxos Monoinsaturados/química , Hematócrito , Contagem de Leucócitos , Lipídeos/análise , Lipídeos/sangue , Fígado/anatomia & histologia , Fígado/química , Masculino , Tamanho do Órgão , Óleo de Brassica napus , Ratos , Ratos Sprague-DawleyRESUMO
Previous research in rats and mice has suggested that gamma-linolenic acid (GLA) derived from borage oil (BO: 23% GLA) may be an appropriate source for increasing levels of long-chain n-6 FA in the developing brain. Recently, transgenic technology has made available a highly enriched GLA seed oil from the canola plant (HGCO: 36% GLA). The first objective of this study was to compare the effects of diets containing equal levels of GLA (23%) from either BO or HGCO on reproduction, pup development, and pup brain FA composition in mice. The second objective was to compare the effects of the HGCO diluted to 23% GLA (GLA-23) with those of undiluted HGCO containing 36% GLA (GLA-36). The diets were fed to the dams prior to conception and throughout pregnancy and lactation, as well as to the pups after weaning. The behavioral development of the pups was measured 12 d after birth, and anxiety in the adult male offspring was assessed using the plus maze. The findings show that despite equivalent levels of GLA, GLA-23 differed from BO in that it reduced pup body weight and was associated with a slight increase in neonatal pup attrition. However, there were no significant effects on pup behavioral development or on performance in the plus maze. An increase in dietary GLA resulted in an increase in brain 20:4n-6 and 22:4n-6, with a corresponding decrease in 22:6n-3. Again, despite their similar levels of GLA, these effects tended to be larger in GLA-23 than in BO. In comparison with GLA-23, GLA-36 had larger effects on growth and brain FA composition but no differences with respect to effects on reproduction and behavioral development. These findings suggest that the HGCO can be used as an alternative source of GLA.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Crescimento/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Ácido gama-Linolênico/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Ácidos Graxos/análise , Feminino , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Óleos de Plantas/farmacologia , Gravidez , Óleo de Brassica napusRESUMO
In mammalian cells, Sprecher has proposed that the synthesis of long-chain PUFA from the 20-carbon substrates involves two consecutive elongation steps, a delta6-desaturation step followed by retroconversion (Sprecher, H., Biochim. Biophys. Acta 1486, 219-231, 2000). We searched the database using the translated sequence of human elongase ELOVL5, whose encoded enzyme elongates monounsaturated and polyunsaturated FA, as a query to identify the enzyme(s) involved in elongation of very long chain PUFA. The database search led to the isolation of two cDNA clones from human and mouse. These clones displayed deduced amino acid sequences that had 56.4 and 58% identity, respectively, to that of ELOVL5. The open reading frame of the human clone (ELOVL2) encodes a 296-amino acid peptide, whereas the mouse clone (Elovl2) encodes a 292-amino acid peptide. Expression of these open reading frames in baker's yeast, Saccharomyces cerevisiae, demonstrated that the encoded proteins were involved in the elongation of both 20- and 22-carbon long-chain PUFA, as determined by the conversion of 20:4n-6 to 22:4n-6, 22:4n-6 to 24:4n-6, 20:5n-3 to 22:5n-3, and 22:5n-3 to 24:5n-3. The elongation activity of the mouse Elovl2 was further demonstrated in the transformed mouse L cells incubated with long-chain (C20- and C22-carbon) n-6 and n-3 PUFA substrates by the significant increase in the levels of 24:4n-6 and 24:5n-3, respectively. This report demonstrates the isolation and identification of two mammalian genes that encode very long chain PUFA specific elongation enzymes in the Sprecher pathway for DHA synthesis.
Assuntos
Acetiltransferases/genética , Acetiltransferases/metabolismo , Ácidos Graxos Ômega-3/biossíntese , Ácidos Graxos Insaturados/biossíntese , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , DNA Complementar/genética , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos , Ácidos Graxos Ômega-6 , Cromatografia Gasosa-Espectrometria de Massas , Vetores Genéticos/genética , Humanos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Especificidade de Órgãos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Five plant-based weaning foods (WF) (Dietrend, Jot-M, Soy, Ang, and Vic-T) locally prepared in Jos, Nigeria were analyzed by gas-liquid chromatography, reverse-phase high performance liquid chromatography, and atomic emission spectrometry with inductively coupled plasma to determine their fatty acid (FA), amino acid, and trace mineral contents, respectively. Results of these direct analyses were compared to expected values derived from food composition tables prepared by the United States Department of Agriculture (USDA). Additionally, results were compared against recommended nutrient values, using breast milk as the standard for FA content and recommended dietary allowances (RDA) for amino acid and mineral contents. The overall nutritional value of the five WF varied considerably and the quantities of particular nutrients determined by direct analysis differed markedly from those estimated using USDA food tables. Comparison of WF fatty acid composition relative to the RDA recommendations and a human milk standard revealed a much higher proportion of both linoleic (35-55 wt%) and alpha-linolenic acids (1%-7 wt%) relative to human milk lipids (11%-12% and 0.8%-0.9% wt, respectively); however, the WF were devoid of arachidonic acid and docosahexaenoic acid. Soy contained the highest amounts of linoleic acid (59.7 mg/g) and alpha-linolenic acid (7.46 mg/g) compared to the other four WF (10.2-41.0 and 0.35-3.18 mg/g, respectively). The linoleic acid/alpha-linolenic acid ratio was within the recommended range (5:1 to 10:1) in only Jot-M (10:1) and Soy (8:1). Dietrend, Vic-T and Ang, contained linoleic/alpha-linolenic ratios of 12:1, 29:1, and 82:1, respectively. The Soy weaning food would provide the most protein (24.3 g/day), based on an estimated daily intake of 65 g of weaning food by a normal six-month-old infant, compared to Jot-M (11.9 g/day), Dietrend (11.7 g/day), Ang (8.07 g/day), and Vic-T (7.26 g/day). The protein RDA for children up to 1 year of age is 13-14 g/day. Comparison of the mineral contents of the WF to the RDAs for various minerals indicated that all five would provide suboptimal amounts of calcium (16 to 250 mg/day) and zinc (1.42 to 3.56 mg/day) compared to respective RDAs of 400 mg/day and 5 mg/day. These data show that the Soy weaning food is an excellent source of linoleic acid and alpha-linolenic acid, as well as being a good source of high quality protein. Jot-M and Dietrend provide useful amounts of the essential FA; however, it is advisable to reevaluate the composition of Ang and Vic-T to find ways to improve the linoleic/alpha-linolenic ratio of each and increase their total protein content. These results document the shortcomings of using published food composition tables based on foods in America when devising weaning foods based on ingredients in another part of the world.