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1.
J Agric Food Chem ; 71(38): 14068-14078, 2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37679308

RESUMO

Bovine ß-lactoglobulin (BLG) is a common allergen found in milk, and the immunoglobulin E (IgE) epitope plays a crucial role in cow milk allergy. Therefore, targeting the IgE epitope could be useful in accurately detecting BLG and assessing its allergenicity. However, producing an IgE epitope-specific antibody (IgE-EsAb) through traditional methods requires complex and time-consuming procedures. Here, IgE-EsAb was purified from rabbit anti-BLG sera by immunomagnetic beads in one step. Then, a sandwich ELISA (sELISA) based on the IgE-EsAb was developed to detect BLG and predict the potential milk allergenicity in foods. The obtained IgE-EsAb could specifically recognize the target IgE epitope of BLG and exhibited high affinity and specificity. The developed IgE-EsAb-based sELISA demonstrated an ultra-wide linear range of 3.9-1.28 × 105 ng/mL, with a limit of detection of 0.49 ng/mL for BLG. Additionally, the proposed immunoassay showed high specificity and recoveries (91.24-109.61%). The ability of the IgE-EsAb-based sELISA to evaluate the potential milk allergenicity in foods was validated using sera from cow milk allergy patients. These results suggest that immunomagnetic beads are an effective tool for rapidly obtaining the IgE-EsAb, and our proposed sELISA could be a reliable and user-friendly method for monitoring trace amounts of BLG and predicting the potential milk allergenicity of food samples.


Assuntos
Alérgenos , Hipersensibilidade a Leite , Feminino , Humanos , Bovinos , Animais , Coelhos , Epitopos , Lactoglobulinas/análise , Imunoglobulina E
3.
Sci Rep ; 8(1): 4809, 2018 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-29556076

RESUMO

Excessive triglyceride accumulation in hepatocytes is the hallmark of obesity-associated nonalcoholic fatty liver disease (NAFLD). Elevated levels of the saturated free fatty acid palmitate in obesity are a major contributor to excessive hepatic lipid accumulation. The nuclear orphan receptor Nur77 is a transcriptional regulator and a lipotoxicity sensor. Using human HepG2 hepatoma cells, this study aimed to investigate the functional role of Nur77 in palmitate-induced hepatic steatosis. The results revealed that palmitate significantly induced lipid accumulation and suppressed lipolysis in hepatocytes. In addition, palmitate significantly suppressed Nur77 expression and stimulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes. Nur77 overexpression significantly reduced palmitate-induced expression of PPARγ and its target genes. Moreover, Nur77 overexpression attenuated lipid accumulation and augmented lipolysis in palmitate-treated hepatocytes. Importantly, G0S2 knockdown significantly attenuated lipid accumulation and augmented lipolysis in palmitate-treated hepatocytes, whereas G0S2 knockdown had no effect on the palmitate-induced expression of Nur77, PPARγ, or PPARγ target genes. In summary, palmitate suppresses Nur77 expression in HepG2 cells, and Nur77 overexpression alleviates palmitate-induced hepatic fat accumulation by down-regulating G0S2. These results display a novel molecular mechanism linking Nur77-regulated G0S2 expression to palmitate-induced hepatic steatosis.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metabolismo dos Lipídeos , Lipólise , Neoplasias Hepáticas/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Palmitatos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia
4.
World J Gastroenterol ; 23(43): 7705-7715, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29209111

RESUMO

AIM: To determine the role of G0/G1 switch gene 2 (G0S2) and its transcriptional regulation in palmitate-induced hepatic lipid accumulation. METHODS: HepG2 cells were treated with palmitate, or palmitate in combination with CCAAT/enhancer binding protein (C/EBP)ß siRNA or G0S2 siRNA. The mRNA expression of C/EBPß, peroxisome proliferator-activated receptor (PPAR)γ and PPARγ target genes (G0S2, GPR81, GPR109A and Adipoq) was examined by qPCR. The protein expression of C/EBPß, PPARγ, and G0S2 was determined by Western blotting. Lipid accumulation was detected with Oil Red O staining and quantified by absorbance value of the extracted Oil Red O dye. Lipolysis was evaluated by measuring the amount of glycerol released into the medium. RESULTS: Palmitate caused a dose-dependent increase in lipid accumulation and a dose-dependent decrease in lipolysis in HepG2 cells. In addition, palmitate increased the mRNA expression of C/EBPß, PPARγ, and PPARγ target genes (G0S2, GPR81, GPR109A, and Adipoq) and the protein expression of C/EBPß, PPARγ, and G0S2 in a dose-dependent manner. Knockdown of C/EBPß decreased palmitate-induced PPARγ and its target genes (G0S2, GPR81, GPR109A, and Adipoq) mRNA expression and palmitate-induced PPARγ and G0S2 protein expression in HepG2 cells. Knockdown of C/EBPß also attenuated lipid accumulation and augmented lipolysis in palmitate-treated HepG2 cells. G0S2 knockdown attenuated lipid accumulation and augmented lipolysis, while G0S2 knockdown had no effects on the mRNA expression of C/EBPß, PPARγ, and PPARγ target genes (GPR81, GPR109A and Adipoq) in palmitate-treated HepG2 cells. CONCLUSION: Palmitate can induce lipid accumulation in HepG2 cells by activating C/EBPß-mediated G0S2 expression.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metabolismo dos Lipídeos/fisiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Palmitatos/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas de Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lipólise/fisiologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR gama/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo
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