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1.
Early Hum Dev ; 186: 105873, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37844515

RESUMO

OBJECTIVE: To compare the neonatal outcomes of early preterm births according to delivery indications and determine the obstetric risk factors associated with adverse outcomes. METHODS: We retrospectively studied pregnancies delivered between 22 + 0 and 26 + 6 weeks at the tertiary center between April 2013 and April 2022. Stillbirths, elective termination of pregnancy, and multifetal pregnancies were excluded. Patients were classified into two groups according to delivery indications: spontaneous preterm birth (sPTB) due to premature rupture of membranes (PROM), preterm labor, or acute cervical insufficiency; and indicated preterm birth (iPTB). Obstetric and neonatal outcomes were compared between the groups. RESULTS: Of the 121 neonates, 73 % (88/121) underwent sPTB. The overall survival rates were 73 % and 49 % in the sPTB and iPTB groups, respectively (p = 0.017). Multivariate logistic regression analysis was performed with adjustment for gestational age at delivery, fetal growth restriction, cesarean section, histological chorioamnionitis, and funisitis. Moreover, in the 1-year follow-up, the proportion of body mass below the third percentile was significantly higher in the iPTB-group than in the sPTB-group (53 % vs. 20 %, p = 0.019). Furthermore, diagnoses of developmental delay and cerebral palsy were slightly higher in the iPTB-group (33 % and 20 %, respectively) than in the sPTB-group (27 % and 9 %, respectively); however, this difference was not statistically significant. CONCLUSIONS: In early preterm births, iPTB was associated with a higher neonatal mortality than sPTB.


Assuntos
Corioamnionite , Trabalho de Parto Prematuro , Nascimento Prematuro , Humanos , Gravidez , Recém-Nascido , Feminino , Nascimento Prematuro/epidemiologia , Estudos Retrospectivos , Cesárea , Idade Gestacional
2.
Plants (Basel) ; 12(4)2023 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-36840106

RESUMO

Event DS rice producing protopanaxadiol (PPD) has been previously developed by inserting Panax ginseng dammarenediol-II synthase gene (PgDDS) and PPD synthase gene (CYP716A47). We performed a gas chromatography-mass spectrometry (GC-MS)-based metabolomics of the DS rice to identify metabolic alterations as the effects of genetic engineering by measuring the contents of 65 metabolites in seeds and 63 metabolites in leaves. Multivariate analysis and one-way analysis of variance between DS and non-genetically modified (GM) rice showed that DS rice accumulated fewer tocotrienols, tocopherols, and phytosterols than non-GM rice. These results may be due to competition for the same precursors because PPDs in DS rice are synthesized from the same precursors as those of phytosterols. In addition, multivariate analysis of metabolic data from rice leaves revealed that composition differed by growth stage rather than genetic modifications. Our results demonstrate the potential of metabolomics for identifying metabolic alterations in response to genetic modifications.

3.
J Pharm Biomed Anal ; 179: 112987, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31761376

RESUMO

Motolimod (VTX-2337) is an agonist of toll-like receptor 8. In this study, a novel and sensitive liquid chromatography-tandem mass spectrometry method was developed for quantifying motolimod in rat plasma and subsequently used in a pharmacokinetic study. Proteins were precipitated from plasma samples using acetonitrile prior to the analysis. GS-9620 was used as an internal standard. High-performance liquid chromatography was performed using a Spursil C18-EP column (3 µm, 50 × 2.1 mm). Aqueous ammonium formate and acetonitrile were used as the mobile phase. Motolimod was detected using an electrospray ionization interface under multiple reaction monitoring conditions in the positive ion mode. The developed method produced a linear correlation over a concentration range of 1-1000 ng/mL (r = 0.9944). Intra- and inter-day precision values ranged from 4.8%-10.7% (the lower limit of quantification precision value was 16.3 %), whereas intra- and inter-day accuracy values ranged from 0.3%-9.1 %. The mean recovery of motolimod from rat plasma was 109.4 %. Additionally, motolimod was found to be stable under various conditions (three freeze-thaw cycles, 6-h storage at room temperature, short- and long-term stability tests, and processing). The developed method was successfully used in a pharmacokinetic study in rats. Motolimod showed non-linear pharmacokinetics following its intravenous administration to rats at 0.6-6 mg/kg. Additionally, very low exposure (<1 %) was obtained following oral administration of the drug to rats. The results also showed that motolimod has a low metabolic stability in the liver microsomes and exhibits extensive binding to the plasma proteins.


Assuntos
Benzazepinas/química , Benzazepinas/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Benzazepinas/sangue , Estabilidade de Medicamentos , Limite de Detecção , Masculino , Microssomos Hepáticos/metabolismo , Ligação Proteica , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodos
4.
Chem Commun (Camb) ; 55(70): 10424-10427, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31407744

RESUMO

The development of sensitive and reliable fluorescent probes for the early diagnosis of Alzheimer's disease (AD) is highly challenging and plays an important role in achieving effective treatments. Herein, we designed and synthesized an indole-based fluorophore for TTR in human plasma, an important hallmark of AD pathogenesis. This robust and simple fluorescent method allows quantification of TTR in the complex biological matrix.


Assuntos
Doença de Alzheimer/diagnóstico , Corantes Fluorescentes/química , Pré-Albumina/metabolismo , Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/metabolismo , Humanos , Limite de Detecção , Espectrometria de Fluorescência
5.
J Pharm Biomed Anal ; 164: 590-597, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30469108

RESUMO

The antioxidant enzyme human extracellular superoxide dismutase (SOD3) is a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry procedure was developed and validated for the determination of SOD3 levels in the plasma of ICR mice. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C18 column (2.1 × 50 mm, 3.5 µm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. The calibration curves were linear (r > 0.99) between 5 and 1000 µg/mL with a lower limit of quantification of 5 µg/mL. The relative standard deviation ranged from 3.08 to 8.84% while the relative error ranged from -0.13 to -9.56%. This method was successfully applied to a preclinical pharmacokinetic study of SOD3 in male ICR mice.


Assuntos
Produtos Biológicos/farmacocinética , Fracionamento Químico/métodos , Superóxido Dismutase/farmacocinética , Animais , Produtos Biológicos/sangue , Fracionamento Químico/instrumentação , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Células HEK293 , Humanos , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Animais , Peptídeos/sangue , Peptídeos/isolamento & purificação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacocinética , Padrões de Referência , Reprodutibilidade dos Testes , Organismos Livres de Patógenos Específicos , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Superóxido Dismutase/administração & dosagem , Superóxido Dismutase/sangue , Superóxido Dismutase/isolamento & purificação , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
6.
Analyst ; 141(8): 2481-6, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-26980003

RESUMO

Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.


Assuntos
Técnicas Biossensoriais/métodos , Toxinas Botulínicas/análise , Endopeptidases/metabolismo , Exopeptidases/metabolismo , Testes Imediatos , Adsorção , Sequência de Aminoácidos , Aminofenóis/química , Técnicas Biossensoriais/instrumentação , Toxinas Botulínicas/química , Toxinas Botulínicas/metabolismo , Ditiotreitol/química , Eletroquímica , Eletrodos , Limite de Detecção , Proteólise , Compostos de Estanho/química
7.
Genes Dev ; 29(15): 1599-604, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26253535

RESUMO

Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (Kd = ∼ 6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2-let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing.


Assuntos
Proteínas Argonautas/metabolismo , Inativação Gênica/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , MicroRNAs/metabolismo , Animais , Células Cultivadas , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Camundongos , Ligação Proteica , Estabilidade de RNA/fisiologia
8.
Biosens Bioelectron ; 72: 211-7, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25982730

RESUMO

Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues.


Assuntos
Toxinas Botulínicas/análise , Clostridium botulinum/enzimologia , Técnicas Eletroquímicas/métodos , Neurotoxinas/análise , Técnicas Biossensoriais/métodos , Toxinas Botulínicas/metabolismo , Botulismo/microbiologia , Clostridium botulinum/isolamento & purificação , Clostridium botulinum/metabolismo , Água Potável/microbiologia , Humanos , Limite de Detecção , Neurotoxinas/metabolismo , Oligopeptídeos/metabolismo , Oxirredução , Proteólise
9.
Oncol Rep ; 33(3): 1526-32, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25592775

RESUMO

2,4,6,8-(3)-Tetranitrophenyl-3,7-diazabicyclo[3.3.1]nonan-9-one (B16), a bispidinone analog, was synthesized to investigate its effects on cell viability, the cell cycle, and apoptotic pathways in HeLa human cervical cancer cells. B16 decreased the percentage of viable cells in WST-8 assays, and morphological changes associated with apoptotic cell death were observed, including cell shrinkage and disruption. Annexin V-FITC/PI dual staining assays showed that B16 significantly increased the early apoptosis of HeLa cells after 24 h of treatment. Moreover, DNA content analysis and [3H]-thymidine incorporation assays showed that B16 induced S-phase cell cycle arrest and inhibited DNA replication after 24 h of treatment. Following treatment with 25 µM of B16, an increase in reactive oxygen species and a decrease in mitochondrial membrane potential were observed by flow cytometry. In addition, the expression levels of caspase cascade and Bcl-2 family proteins determined by western blotting suggested that the induction of apoptosis by B16 was associated with a caspase- and mitochondrial-dependent pathway in HeLa cells. In conclusion, B16 induced early apoptosis and S-phase cell cycle arrest in HeLa cells via a caspase- and mitochondrial­dependent pathway.


Assuntos
Apoptose/efeitos dos fármacos , Compostos Azabicíclicos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nitrobenzenos/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Compostos Aza/química , Compostos Aza/farmacologia , Compostos Azabicíclicos/síntese química , Caspases/biossíntese , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Nitrobenzenos/síntese química , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Espécies Reativas de Oxigênio/metabolismo
10.
Mol Biol Rep ; 40(1): 269-79, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23054007

RESUMO

Ginsenoside Rg3 is one of ginsenosides that are the well-known bioactive principles of Panax ginseng. Among the two stereoisomeric forms of Rg3, 20(S)-ginsenoside Rg3 [20(S)-Rg3] is predominant. 20(S)-Rg3 is capable of suppressing the nitric oxide (NO), reactive oxygen species (ROS) and prostaglandin E2 (PGE2) productions induced by lipopolysaccharide (LPS) in RAW264.7 macrophage cells in a concentration-dependent manner. In the same stimulated macrophages, 20(S)-Rg3 was able to suppress matrix metalloproteinase-9 (MMP-9) activity and suppress cyclooxygenase-2 (COX-2) expression. It suppressed the production of some proinflammatory cytokines, such as TNF-α, IL-1ß and IL-6, and the cell mobility enhanced by LPS in the macrophage cells. 20(S)-Rg3 displayed suppressive effect on the ROS level but not on the NO level, and down-regulating effect on MMP-9 but not on MMP-2 in non-stimulated HaCat keratinocytes. 20(S)-Rg3 also exhibited suppressive effect on the MMP-9 gelatinolytic activity enhanced in the HaCat keratinocytes stimulated with tumor necrosis factor-α (TNF-α), one of the major proinflammatory cytokines. However, 20(S)-Rg3 was not able to modulate the NO level even in the presence of TNF-α. Taken together, anti-inflammatory and related antioxidative and MMP-9 inhibitory activities of 20(S)-Rg3, the major stereoisomeric form of ginsenoside Rg3, are confirmed in macrophage and keratinocyte cell lines.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Ginsenosídeos/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Animais , Anti-Inflamatórios/química , Antioxidantes/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Dinoprostona/biossíntese , Ginsenosídeos/química , Humanos , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Óxido Nítrico/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
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