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1.
Food Res Int ; 161: 111859, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36192983

RESUMO

Poultry products are an essential animal source of protein for humans. Many factors could destroy the balance of the poultry production chain and cause an overstock of products, which need to be stored in the frozen storage warehouse for a long time. The long-term frozen storage may affect the quality of meat products. In this study, the changes of small molecular substances were revealed in duck meat during long-term storage using non-targeted metabolomics. The results showed that compared with fresh meat, even if the meat is stored under frozen storage conditions, the number of differential metabolites of frozen storage meat continues to increase with the prolongation of storage time, indicating that the meat composition has changed significantly with the storage time increased. With the increase in storage time, the nitrogen-containing small molecular compounds in duck meat increased (carnosine and anserine, aspartic acid, and tyrosine, 1H-indole-3-acetamide, 2-Hydroxyphenethylamine, 2-Naphylamine, allocystathionine, and O-phosphoethanolamine), the nucleotides decomposition process strengthened (IMP and AMP, GMP and UMP), and the content of organic acid increased (5-hydroxy indole acetic acid, 5-hydroxypentanoic acid and phenylacetate, taurine) and carbohydrate (1-O-sinapoyl-beta-d-glucose, 4-O-beta-d-glucopyranosyl-d-mannose, and alpha-d-glucose). These small molecular substances can be used as biomarkers to detect long-term stored duck meat deterioration. KEGG enrichment analysis showed that protein catabolism, nucleotide catabolism, fat decomposition and oxidation, and carbohydrate decomposition were the main metabolic processes of meat deterioration during the long-term storage of duck meat. In addition, Non-target metabolome technology is a powerful tool to reveal the meat deterioration process during long-term storage systematically. This study provided a reference for optimizing domestic poultry meat storage methods and ensuring food safety.


Assuntos
2-Hidroxifenetilamina , Carnosina , 2-Hidroxifenetilamina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Anserina/metabolismo , Ácido Aspártico/metabolismo , Carboidratos , Carnosina/metabolismo , Patos/metabolismo , Glucose/metabolismo , Humanos , Inosina Monofosfato/metabolismo , Carne/análise , Nitrogênio/metabolismo , Fenilacetatos/metabolismo , Taurina/metabolismo , Tirosina/metabolismo , Uridina Monofosfato/metabolismo
2.
Anal Methods ; 13(10): 1278-1285, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33624658

RESUMO

In the present study, an antibody against phenylethanolamine A (PEA) was produced, confirmed, and used in a surface plasmon resonance (SPR)-based measurement. Bovine serum albumin (BSA)-conjugated PEA was linked to nano-gold particles bound to l-cysteine modified on the surface of a Au-NP sensor chip. The concentrations of antigen and antibody were optimized, and the designed biosensor chip was investigated to examine the stability and accuracy of the proposed method. The recovery of PEA ranged from 80.4-93.4% in swine urine samples with spike levels of 5, 10 and 20 ng mL-1, and the relative standard deviations of PEA were less than 2%. PEA analogues, such as clenbuterol, ractopamine, and salbutamol, did not influence the PEA measurement. The developed method could be used to measure PEA in swine urine samples.


Assuntos
2-Hidroxifenetilamina , Técnicas Biossensoriais , 2-Hidroxifenetilamina/análogos & derivados , Animais , Ouro , Ressonância de Plasmônio de Superfície , Suínos
3.
Clin Cancer Res ; 26(18): 4777-4784, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32616501

RESUMO

PURPOSE: AT13148 is an oral AGC kinase inhibitor, which potently inhibits ROCK and AKT kinases. In preclinical models, AT13148 has been shown to have antimetastatic and antiproliferative activity. PATIENTS AND METHODS: The trial followed a rolling six design during dose escalation. An intrapatient dose escalation arm to evaluate tolerability and a biopsy cohort to study pharmacodynamic effects were later added. AT13148 was administered orally three days a week (Mon-Wed-Fri) in 28-day cycles. Pharmacokinetic profiles were assessed using mass spectrometry and pharmacodynamic studies included quantifying p-GSK3ß levels in platelet-rich plasma (PRP) and p-cofilin and p-MLC2 levels in tumor biopsies. RESULTS: Fifty-one patients were treated on study. The safety of 5-300 mg of AT13148 was studied. Further, the doses of 120-180-240 mg were studied in an intrapatient dose escalation cohort. The dose-limiting toxicities included hypotension (300 mg), pneumonitis, and elevated liver enzymes (240 mg), and skin rash (180 mg). The most common side effects were fatigue, nausea, headaches, and hypotension. On the basis of tolerability, 180 mg was considered the maximally tolerated dose. At 180 mg, mean C max and AUC were 400 nmol/L and 13,000 nmol/L/hour, respectively. At 180 mg, ≥50% reduction of p-cofilin was observed in 3 of 8 posttreatment biopsies. CONCLUSIONS: AT13148 was the first dual potent ROCK-AKT inhibitor to be investigated for the treatment of solid tumors. The narrow therapeutic index and the pharmacokinetic profile led to recommend not developing this compound further. There are significant lessons learned in designing and testing agents that simultaneously inhibit multiple kinases including AGC kinases in cancer.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Antineoplásicos/efeitos adversos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/efeitos adversos , 2-Hidroxifenetilamina/administração & dosagem , 2-Hidroxifenetilamina/efeitos adversos , 2-Hidroxifenetilamina/farmacocinética , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Relação Dose-Resposta a Droga , Erupção por Droga/epidemiologia , Erupção por Droga/etiologia , Feminino , Cefaleia/induzido quimicamente , Cefaleia/epidemiologia , Humanos , Hiperglicemia/induzido quimicamente , Hiperglicemia/epidemiologia , Hipotensão/induzido quimicamente , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/sangue , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirazóis/administração & dosagem , Pirazóis/farmacocinética , Quinases Associadas a rho/antagonistas & inibidores
4.
Metabolomics ; 16(4): 50, 2020 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-32285223

RESUMO

INTRODUCTION: To generate biomarkers of target engagement or predictive response for multi-target drugs is challenging. One such compound is the multi-AGC kinase inhibitor AT13148. Metabolic signatures of selective signal transduction inhibitors identified in preclinical models have previously been confirmed in early clinical studies. This study explores whether metabolic signatures could be used as biomarkers for the multi-AGC kinase inhibitor AT13148. OBJECTIVES: To identify metabolomic changes of biomarkers of multi-AGC kinase inhibitor AT13148 in cells, xenograft / mouse models and in patients in a Phase I clinical study. METHODS: HILIC LC-MS/MS methods and Biocrates AbsoluteIDQ™ p180 kit were used for targeted metabolomics; followed by multivariate data analysis in SIMCA and statistical analysis in Graphpad. Metaboanalyst and String were used for network analysis. RESULTS: BT474 and PC3 cells treated with AT13148 affected metabolites which are in a gene protein metabolite network associated with Nitric oxide synthases (NOS). In mice bearing the human tumour xenografts BT474 and PC3, AT13148 treatment did not produce a common robust tumour specific metabolite change. However, AT13148 treatment of non-tumour bearing mice revealed 45 metabolites that were different from non-treated mice. These changes were also observed in patients at doses where biomarker modulation was observed. Further network analysis of these metabolites indicated enrichment for genes associated with the NOS pathway. The impact of AT13148 on the metabolite changes and the involvement of NOS-AT13148- Asymmetric dimethylarginine (ADMA) interaction were consistent with hypotension observed in patients in higher dose cohorts (160-300 mg). CONCLUSION: AT13148 affects metabolites associated with NOS in cells, mice and patients which is consistent with the clinical dose-limiting hypotension.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Antineoplásicos/metabolismo , Metabolômica , Óxido Nítrico Sintase/antagonistas & inibidores , Inibidores de Proteínas Quinases/metabolismo , Pirazóis/metabolismo , 2-Hidroxifenetilamina/administração & dosagem , 2-Hidroxifenetilamina/metabolismo , 2-Hidroxifenetilamina/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Biomarcadores Tumorais/sangue , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Glicogênio Sintase Quinase 3 beta/sangue , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Óxido Nítrico Sintase/metabolismo , Células PC-3 , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/administração & dosagem , Pirazóis/farmacologia
5.
Cancer Res ; 78(12): 3321-3336, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29669760

RESUMO

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries.Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321-36. ©2018 AACR.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 2-Hidroxifenetilamina/farmacologia , 2-Hidroxifenetilamina/uso terapêutico , Amidas/farmacologia , Amidas/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral/transplante , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/metabolismo
6.
Biosens Bioelectron ; 99: 21-27, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-28732345

RESUMO

Herein, we reported a disposable electroanalytical device for competitive enzyme-linked immunosorbent assay (ELISA) of phenylethanolamine A (PA). Conductive carbon tape coated with gold cluster served as the working electrode, while the counter and reference electrodes were fabricated on the filter paper by the screen-printing technique. Separating fabrication of the working electrode from other electrodes could make it possible for full utilization of the working electrode modification in bulk and no contamination of the immune-reagents on the counter and reference electrodes. The gold cluster played an important role in both immobilizing antigen and accelerating electron transfer. The eight-channel devices were utilized to detect PA on the strategy of competitive ELISA. The detection range and the limit of detection (LOD) using differential pulse voltammetry for PA were 0.005-60ngmL-1 and 2.6pgmL-1, respectively. And also, the linear range for PA performed by square wave voltammetry was 0.05-60ngmL-1 with the LOD value as 0.028ngmL-1. The results clearly demonstrated that the proposed electroanalytical devices could be successfully applied in immunoassay and might be further developed for determination of different analytes based on ELISA format.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Técnicas Biossensoriais , Ensaio de Imunoadsorção Enzimática , 2-Hidroxifenetilamina/isolamento & purificação , Carbono/química , Eletroquímica , Ouro/química , Limite de Detecção
7.
J Anal Toxicol ; 41(2): 146-152, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-27881619

RESUMO

The present study proposed the use of liquid chromatography-tandem mass spectrometry to detect the novel ß-agonist phenylethanolamine A (PEA) in incurred hair samples of swine and sheep and to assess its accumulation for residue monitoring. The method showed good percent recoveries ranging from 93.2% to 102% and good coefficient of variation at <15%. The experiment was conducted in swine (24 treated and 4 controls) and sheep (3 treated and 1 control). PEA concentration was determined in hair during the treatment. High residue concentrations were present in hair as early as Day 24 (14.8 ± 3.6 and 25.8 ± 7.6 ng/g) and Day 21 (23.4 ± 6.6 ng/g) for swine and sheep; these residues persisted until withdrawal on Days 14 and 21. Results showed high PEA accumulation in hair, thereby indicating the use of hair as a matrix in the control of PEA abuse in farm animals.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Agonistas Adrenérgicos beta/análise , Resíduos de Drogas/análise , Cabelo/química , Detecção do Abuso de Substâncias/métodos , Detecção do Abuso de Substâncias/veterinária , 2-Hidroxifenetilamina/análise , Animais , Cromatografia Líquida , Feminino , Masculino , Ovinos , Detecção do Abuso de Substâncias/instrumentação , Suínos , Espectrometria de Massas em Tandem
8.
J Sci Food Agric ; 97(3): 1001-1009, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27247162

RESUMO

BACKGROUND: All ß-agonists are banned as feed additives for growth promotion in animals due to toxic effects on humans after consuming the ß-agonist contaminated meats. Phenylethanolamine A (PA) is a newly emerged ß-agonist. Thus there is a need to develop highly sensitive and specific analytical methods for the detection of PA in food samples. In this study, the monoclonal antibody (mAb) against PA was produced by hybridoma technology and used for the development of enzyme-linked immunosorbent assay (ELISA). RESULTS: The IC50 values and limits of detection (LODs) of the ELISA using homogeneous combination of coating antigen/antibody for PA were 0.16 ng mL-1 and 0.011 ng mL-1 , respectively. The cross-reactive (CR) values of the assay with 14 structurally related ß-agonists were lower than 0.59%. Swine liver and meat samples were spiked with PA at different content and analysed by ELISA. Acceptable recovery rates of 91.40-105.51% and intra-assay coefficients of variation of 1.56-9.92% (n = 3) were obtained. The ELISA for seven spiked samples was confirmed by LC-MS/MS with a high correlation coefficient of 0.9881. CONCLUSION: The proposed mAb-based ELISA was highly sensitive and specific for PA and could be used as a quantitative/screening method for PA analysis in food samples. © 2016 Society of Chemical Industry.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Agonistas Adrenérgicos beta/análise , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Resíduos de Drogas/análise , Ensaio de Imunoadsorção Enzimática , Inspeção de Alimentos/métodos , 2-Hidroxifenetilamina/análise , 2-Hidroxifenetilamina/química , 2-Hidroxifenetilamina/metabolismo , Agonistas Adrenérgicos beta/química , Agonistas Adrenérgicos beta/metabolismo , Animais , China , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Resíduos de Drogas/química , Resíduos de Drogas/metabolismo , Contaminação de Alimentos , Haptenos/química , Haptenos/metabolismo , Limite de Detecção , Fígado/química , Carne/análise , Estrutura Molecular , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Sus scrofa , Espectrometria de Massas em Tandem
9.
Neuron ; 92(6): 1324-1336, 2016 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-27916457

RESUMO

Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino-terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete because of the lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc-binding site and reveal distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability, supporting the general model in which the bi-lobe motion in ATD regulates the channel activity in NMDA receptors.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Zinco/metabolismo , 2-Hidroxifenetilamina/metabolismo , Animais , Sítios de Ligação , Western Blotting , Cristalografia , Ligação de Hidrogênio , Piperidinas/farmacologia , Estrutura Quaternária de Proteína , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Células Sf9 , Spodoptera , Xenopus laevis , Zinco/farmacologia
10.
J Labelled Comp Radiopharm ; 59(13): 546-551, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27739098

RESUMO

Three stable and simple synthetic routes of labeled D9 -Mabuterol, D9 -Bambuterol, and D9 -Cimbuterol were described with 98.5%, 99.7%, and 98.4% isotopic abundance and good purity. These structures and isotope-abundance were confirmed according to 1 H NMR and liquid chromatography-tandem mass spectrometry.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Compostos de Anilina/química , Compostos de Anilina/síntese química , Clembuterol/análogos & derivados , Deutério/química , Terbutalina/análogos & derivados , 2-Hidroxifenetilamina/síntese química , 2-Hidroxifenetilamina/química , Técnicas de Química Sintética , Clembuterol/síntese química , Clembuterol/química , Marcação por Isótopo , Terbutalina/síntese química , Terbutalina/química
11.
Biochem Biophys Res Commun ; 478(1): 330-336, 2016 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-26828267

RESUMO

The AGC kinase family is important cell proliferation and survival. Dysregulation of this family contributes to gastric cancer progression. Here, we evaluated the potential activity of AT13148, a first-in-class multi-AGC kinase inhibitor, against gastric cancer cells. Our results showed that AT13148 exerted potent cytotoxic and anti-proliferative activities against a panel human gastric cancer cell lines (HGC-27, AGS, SNU-601, N87 and MKN-28), possibly via inducing cancer cell apoptotic death. Apoptosis inhibition by the Caspase blockers dramatically attenuated AT13148-caused cytotoxicity against gastric cancer cells. Intriguingly, same AT13148 treatment was not cytotoxic/pro-apoptotic to the non-cancerous human gastric epithelial GEC-1 cells. At the signaling level, AT13148 treatment in gastric cancer cells dramatically suppressed activation of multiple AGC kinases, including Akt (at p-Thr-308), p70S6 kinase (p70S6K), glycogen synthase kinase 3ß (GSK-3ß) and p90 ribosomal S6 kinase (RSK). Our in vivo studies demonstrated that daily oral gavage of AT13148 at well-tolerated doses significantly inhibited HGC27 xenograft tumor growth in nude mice. AGC activity was also dramatically decreased in AT13148-administrated HGC27 tumors. Therefore, targeting AGC kinases by AT13148 demonstrates superior anti-gastric cancer activity both in vitro and in vivo. The preclinical results of this study support the progression of this molecule into future evaluation as a valuable anti-gastric cancer candidate.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas Quinases/metabolismo , Pirazóis/administração & dosagem , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , 2-Hidroxifenetilamina/administração & dosagem , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Inibidores de Proteínas Quinases/administração & dosagem , Neoplasias Gástricas/patologia , Resultado do Tratamento
12.
Luminescence ; 31(2): 372-379, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26179292

RESUMO

The interactions of mapenterol with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated systematically using fluorescence spectroscopy, absorption spectroscopy, circular dichroism (CD) and molecular docking techniques. Mapenterol has a strong ability to quench the intrinsic fluorescence of BSA and HSA through static quenching procedures. At 291 K, the binding constants, Ka, were 1.93 × 10(3) and 2.73 × 10(3) L/mol for mapenterol-BSA and mapenterol-HAS, respectively. Electrostatic forces and hydrophobic interactions played important roles in stabilizing the mapenterol-BSA/has complex. Using site marker competitive studies, mapenterol was found to bind at Sudlow site I on BSA/HSA. There was little effect of K(+), Ca(2+), Cu(2+), Zn(2+) and Fe(3+) on the binding. The conformation of BSA/HSA was changed by mapenterol, as seen from the synchronous fluorescence spectra. The CD spectra showed that the binding of mapenterol to BSA/HSA changed the secondary structure of BSA/HSA. Molecular docking further confirmed that mapenterol could bind to Sudlow site I of BSA/HSA. According to Förster non-radiative energy transfer theory (FRET), the distances r0 between the donor and acceptor were calculated as 3.18 and 2.75 nm for mapenterol-BSA and mapenterol-HAS, respectively.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Compostos de Anilina/química , Simulação de Acoplamento Molecular , Albumina Sérica/química , 2-Hidroxifenetilamina/química , Animais , Bovinos , Dicroísmo Circular , Humanos , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
13.
Anal Bioanal Chem ; 407(25): 7615-24, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26255292

RESUMO

Phenylethanolamine A (PA) is a ß-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other ß-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Agonistas Adrenérgicos beta/análise , Agonistas Adrenérgicos beta/urina , Ensaio de Imunoadsorção Enzimática/métodos , Substâncias de Crescimento/análise , Carne Vermelha/análise , Suínos/urina , 2-Hidroxifenetilamina/análise , 2-Hidroxifenetilamina/imunologia , 2-Hidroxifenetilamina/urina , Agonistas Adrenérgicos beta/imunologia , Animais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática/instrumentação , Desenho de Equipamento , Substâncias de Crescimento/urina , Limite de Detecção , Camundongos , Fitas Reagentes/análise
14.
Gene ; 573(1): 153-9, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26187072

RESUMO

OBJECTIVE: To investigate the drug targets related to Notch signaling pathway for glioma treatment. METHODS: Gene expression profiles GSE44561, GSE48079 and GSE22772GSE48079GSE22772 of glioma cells samples with activated Notch signaling pathway and control samples were downloaded from Gene Expression Omnibus database to screen the differentially expressed genes (DEGs) using limma package. GO (Gene Oncology) function and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses were conducted using DAVID tools to predict the underlying function of these DEGs. Sequentially, drug target genes recorded in DrugBank database were collected and matched with the selected DEGs to identify the potential drug targets for glioma. Further, these targets were verified by the screened DEGs in the anti-glioma drug (AT13148) treated samples of microarray data of GSE38008. RESULTS: A total of 75,645,497 DEGs were respectively identified in GSE44561, GSE48079 and GSE22772GSE48079GSE22772 datasets and these DEGs could well distinguish the glioma samples from controls. The DEGs were mainly enriched in classical functions and pathways, such as cell cycle, and DNA replication. A total of 122 DEGs were found to be potential drug targets for glioma, among which GLIPR1 was targeted by drug XL820, PDGFRB and KDR were targeted by SOT-107. Efficacy validation of the other 119 drug targets by GSE38008 data showed that ACSS1, ASL, GCLM, ROCK2, IMPA1, and TFPI may be targeted by the anti-glioma drug of AT13148. CONCLUSION: AT13148 may inhibit glioma progression by suppressing the Notch signaling genes, including GLIPR1, PDGFRB, ACSS1, and ASL.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Antineoplásicos/farmacologia , Neoplasias Encefálicas/patologia , Glioma/patologia , Pirazóis/farmacologia , Receptores Notch/metabolismo , 2-Hidroxifenetilamina/farmacologia , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Humanos
15.
Cancer Res ; 75(11): 2272-84, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25840982

RESUMO

There is an urgent need to identify new therapeutic opportunities for metastatic melanoma. Fragment-based screening has led to the discovery of orally available, ATP-competitive AKT kinase inhibitors, AT13148 and CCT129254. These compounds also inhibit the Rho-kinases ROCK 1 and ROCK 2 and we show they potently inhibit ROCK activity in melanoma cells in culture and in vivo. Treatment of melanoma cells with CCT129254 or AT13148 dramatically reduces cell invasion, impairing both "amoeboid-like" and mesenchymal-like modes of invasion in culture. Intravital imaging shows that CCT129254 or AT13148 treatment reduces the motility of melanoma cells in vivo. CCT129254 inhibits melanoma metastasis when administered 2 days after orthotopic intradermal injection of the cells, or when treatment starts after metastases have arisen. Mechanistically, our data suggest that inhibition of ROCK reduces the ability of melanoma cells to efficiently colonize the lungs. These results suggest that these novel inhibitors of ROCK may be beneficial in the treatment of metastasis.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Movimento Celular/efeitos dos fármacos , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Quinases Associadas a rho/genética , 2-Hidroxifenetilamina/administração & dosagem , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica/genética , Metástase Neoplásica , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores
16.
J Agric Food Chem ; 62(45): 10896-902, 2014 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-25343225

RESUMO

Phenylethanolamine A (PA) is a new kind of ß-agonist, which was illegally used as a feed additive for growth promotion in China. In this study, a novel immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for the ultrasensitive and quantitative detection of phenylethanolamine A is presented. The principle of this new ICA is similar to that based on colloidal gold particles, but using Au(MBA)@Ag-Ab [e.g., polyclonal antibody of PA labeled Au@Ag core-shell nanoparticles (NPs) sandwiched with a Raman reporter (4-mercaptobenzoic acid, MBA)] as a probe. After ICA procedures, the specific Raman scattering intensity of MBA on the test line was measured for quantitative detection of PA. This assay was completed within 15 min. The IC50 and limit of detection (LOD) values of the ICA for PA detection were 0.06 ng mL(-1) and 0.32 pg mL(-1), respectively, which were 1-3 orders of magnitude lower than those obtained by other immunoassays, indicating the ultrasensitivity of this ICA. There was no cross-reactivity (CR) of the assay with another three ß-agonists (ractopamine, clenbuterol, and salbutamol), suggesting high specificity of the SERS-based ICA. A spiking experiment revealed that the recoveries of PA from pig urine samples were in range of 99.9- 101.2% with relative standard deviations (RSDs) of 3.6-5.8%. The results demonstrated that this SERS-based ICA was able to quantitatively detect PA in urine samples with high sensitivity, specificity, precision, and accuracy and might be a powerful method for the analysis of other target analytes in the food area.


Assuntos
2-Hidroxifenetilamina/análise , Agonistas Adrenérgicos beta/análise , Cromatografia de Afinidade/métodos , Análise Espectral Raman/métodos , 2-Hidroxifenetilamina/urina , Agonistas Adrenérgicos beta/urina , Animais , Cromatografia de Afinidade/instrumentação , Ouro/química , Nanopartículas Metálicas/química , Sensibilidade e Especificidade , Suínos
17.
Analyst ; 139(17): 4365-72, 2014 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-25011489

RESUMO

This study reports the development of an electrochemiluminescent (ECL) immunosensor for ultrasensitive detection of phenylethanolamine A (PA) based on CdSe quantum dots (QDs) and gold nanoparticles (GNPs). The GNPs/ovalbumin-PA/anti-PA-QD immunosensor was fabricated layer by layer using GNPs as substrates and electron transport accelerators. The use of GNPs greatly enhanced the sensitivity for detecting PA due to the excellent electron transportation ability and the large surface area of GNP carriers allowing several binding events of ovalbumin-PA on each nanosphere. Transmission electron microscopy images (TEM), photoluminescence spectra, ultraviolet-visible absorption spectra and dynamic light scattering (DLS) were used to characterize the QDs and GNPs. The sensor was characterized with electrochemical impedance spectra (EIS), and a strong ECL emission of the modified electrode could be observed during the cathodic process of S2O8(2-) and QDs in air-saturated PBS buffer containing 0.1 M K2S2O8 and 0.1 M KCl (pH 7.4). With a competitive immunoassay format, the ECL signal depended linearly on the logarithm of the phenylethanolamine A concentration within a range of 0.02 ng mL(-1) to 50 ng mL(-1), and the detection limit was 0.0047 ng mL(-1), much lower than those reported in the literature. This ECL immunosensor is rapid, simple and sensitive with acceptable precision, and it will extend the application of QD ECL in immunoassays of ß-agonists and open new avenues for the detection of food additive residues in the future.


Assuntos
2-Hidroxifenetilamina/análise , Agonistas Adrenérgicos beta/análise , Ouro/química , Imunoensaio/métodos , Medições Luminescentes/métodos , Nanopartículas Metálicas/química , Pontos Quânticos/química , 2-Hidroxifenetilamina/análogos & derivados , 2-Hidroxifenetilamina/urina , Agonistas Adrenérgicos beta/urina , Animais , Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , Compostos de Cádmio/química , Técnicas Eletroquímicas/métodos , Humanos , Limite de Detecção , Carne/análise , Compostos de Selênio/química , Suínos
18.
Eur J Pharmacol ; 723: 62-6, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24275352

RESUMO

The effect of two novel ß3-adrenoceptor (ß3-AR) agonists SP-1f and SP-1h on human colon circular smooth muscle contractility and ß3-AR mRNA expression have been determined. ß3-AR is ascertained co-participates to the control of the gut motility. Isometric tension on human colon muscle strips was measured in response to increasing concentrations of SP-1f, SP-1h and (-)-isoprenaline, alone and in the presence of Betaxolol, ICI 11,855 and SR 59230A (ß1-, ß2- and ß3-AR antagonists, respectively). (-)-Isoprenaline concentration-dependently relaxed circular muscle strips with an EC50=0.32±0.06µM. Such an effect was antagonized either by the contemporaneously presence of Betaxolol and ICI 11,855 [(-)-isoprenaline EC50=1.75±0.35µM, pKB=7.88±0.10] or by Betaxolol, ICI 11,855 and SR 59230A [(-)-isoprenaline EC50=3.49±0.38µM, pKB=8.51±0.14]. Besides, SP-1f and SP-1h concentration-dependently relaxed circular muscle strips with an EC50=0.35±0.07µM and 0.45±0.12µM, respectively. These values remained unchanged by blocking the ß1- and ß2-AR. The presence of SR 59230A antagonized the relaxing effect of SP-1f (EC50=3.51±0.94µM, pKB=8.93±0.16) and did not modify the SP-1h relaxing potency. In colon circular smooth muscle and in mucosa, ß3-AR mRNA expression levels were found to be 0.39±0.70 and 0.26±0.12 (P<0.05), respectively. Such results provide further evidence of the ß3-adrenoceptor functional role in the human colon and the crucial contribution of SP-1f to the control of the gut dysmotility.


Assuntos
2-Hidroxifenetilamina/análogos & derivados , Antagonistas de Receptores Adrenérgicos beta 3/farmacologia , Colo/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Propionatos/farmacologia , Receptores Adrenérgicos beta 3/genética , 2-Hidroxifenetilamina/farmacologia , Idoso , Colo/fisiologia , Feminino , Humanos , Técnicas In Vitro , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Isoproterenol/farmacologia , Masculino , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , RNA Mensageiro/metabolismo , Estereoisomerismo
19.
Electrophoresis ; 34(6): 854-61, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23335131

RESUMO

Dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) was for the first time combined with field-amplified sample injection (FASI) in CE to determine four ß(2)-agonists (cimbuterol, clenbuterol, mabuterol, and mapenterol) in bovine urine. Optimum BGE consisted of 20 mM borate buffer and 0.1 mM SDS. Using salting-out extraction, ß(2)-agonists were extracted into ACN that was then used as the disperser solvent in DLLME-SFO. Optimum DLLME-SFO conditions were: 1.0 mL ACN, 50 µL 1-undecanol (extraction solvent), total extraction time 1.5 min, no salt addition. Back extraction into an aqueous solution (pH 2.0) facilitated direct injection of ß(2)-agonists into CE. Compared to conventional CZE, DLLME-SFO-FASI-CE achieved sensitivity enhancement factors of 41-1046 resulting in LODs in the range of 1.80-37.0 µg L(-1). Linear dynamic ranges of 0.15-10.0 mg L(-1) for cimbuterol and 15-1000 µg L(-1) for the other analytes were obtained with coefficients of determination (R(2)) ≥ 0.9901 and RSD% ≤5.5 (n = 5). Finally, the applicability of the proposed method was successfully confirmed by determination of the four ß(2)-agonists in spiked bovine urine samples and accuracy higher than 96.0% was obtained.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/urina , Eletroforese Capilar/métodos , Microextração em Fase Líquida/métodos , 2-Hidroxifenetilamina/análogos & derivados , 2-Hidroxifenetilamina/urina , Compostos de Anilina/urina , Animais , Bovinos , Clembuterol/análogos & derivados , Clembuterol/urina , Concentração de Íons de Hidrogênio , Sensibilidade e Especificidade , Solventes
20.
Mol Cell Endocrinol ; 371(1-2): 189-94, 2013 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-23267837

RESUMO

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.


Assuntos
Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Feocromocitoma/tratamento farmacológico , Receptores de Neuropeptídeos/efeitos dos fármacos , 2-Hidroxifenetilamina/análogos & derivados , 2-Hidroxifenetilamina/farmacologia , Compostos de Anilina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Camundongos , Pirróis/farmacologia , Receptores LHRH/biossíntese , Receptores LHRH/efeitos dos fármacos , Receptores LHRH/metabolismo , Receptores de Neuropeptídeos/biossíntese , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/biossíntese , Receptores de Hormônios Reguladores de Hormônio Hipofisário/efeitos dos fármacos , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Receptores de Somatostatina/biossíntese , Receptores de Somatostatina/efeitos dos fármacos , Receptores de Somatostatina/metabolismo , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Somatostatina/análogos & derivados
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