RESUMO
To elucidate the potential function of miRNA-221 in gestational diabetes mellitus (GDM) and the underlying mechanism. MiRNA-221 level was analyzed in the microarray containing placental tissues of GDM rats. After constructing GDM model in rats, miRNA-221 level in placental tissues of GDM rats or controls was determined as well. The relationship between miRNA-221 level and blood glucose in GDM rats was analyzed by Spearman correlation test. Regulatory effects of miRNA-221 on proliferation, apoptosis and insulin secretion in INS-1â¯cells were assessed. Through dual-luciferase reporter gene assay, the direct target of miRNA-221, PAK1 was identified. At last, potential influences of miRNA-221/PAK1 axis on INS-1â¯cell phenotypes were determined. MiRNA-221 was downregulated in placental tissues of GDM rats, and its level was negatively correlated to that of blood glucose level in GDM rats. Overexpression of miRNA-221 stimulated insulin secretion, cell proliferation and suppressed apoptosis in INS-1â¯cells. Knockdown of miRNA-221 achieved the opposite results. PAK1 was proved as the direct target of miRNA-221. Notably, PAK1 was able to reverse regulatory effects of miRNA-221 on INS-1â¯cell phenotypes. MiRNA-221 regulates proliferation, apoptosis and insulin secretion in islet ß cells through targeting PAK1, thus protecting GDM-induced islet dysfunction.
Assuntos
Apoptose , Diabetes Gestacional/metabolismo , Células Secretoras de Insulina/citologia , Insulina/metabolismo , MicroRNAs/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Glicemia/metabolismo , Linhagem Celular , Proliferação de Células , Feminino , Teste de Tolerância a Glucose , Células HEK293 , Humanos , Resistência à Insulina , Secreção de Insulina , Gravidez , Prenhez , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
The study of N-linked glycans is among the most challenging bioanalytical tasks because of their complexity and variety. The presence of glycoform families that differ only in branching and/or linkage position makes the identification and quantitation of individual glycans exceedingly difficult. Quantitation of these individual glycans is important because changes in the abundance of these isomers are often associated with significant biomedical events. For instance, previous studies have shown that the ratio of α2-3 to α2-6 linked sialic acid (SA) plays an important role in cancer biology. Consequently, quantitative methods to detect alterations in the ratios of glycans based on their SA linkages could serve as a diagnostic tool in oncology, yet traditional glycomic profiling cannot readily differentiate between these linkage isomers. Here, we present a liquid chromatography-selected reaction monitoring (LC-SRM) approach that we demonstrate is capable of quantitating the individual SA linkage isomers. The LC method is capable of separating sialylated N-glycan isomers differing in α2-3 and α2-6 linkages using a novel superficially porous particle (Fused-Core) Penta-HILIC (hydrophilic interaction liquid chromatography) column. SRM detection provides the relative quantitation of each SA linkage isomer, and minimizes interferences from coeluting glycans that are problematic for UV/Fluorescence based quantitation. With our approach, the relative quantitation of each SA linkage isomer is obtained from a straightforward liquid chromatography-mass spectrometry (LC-MS) experiment.
Assuntos
Cromatografia Líquida/métodos , Fetuínas/química , Polissacarídeos/química , Soro/química , Ácidos Siálicos/análise , Animais , Sequência de Carboidratos , Bovinos , Fetuínas/isolamento & purificação , Humanos , Isomerismo , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Polissacarídeos/isolamento & purificaçãoRESUMO
The chemical compound delta-elemene, isolated from the Chinese herbal medicine plant Curcuma Wenyujin, has been known to exert antitumor activity. In this study we demonstrated that apoptotic cell death induced by delta-elemene in DLD-1 cells was concentration-and time-dependent, and had little inhibition of the normal human liver cell line WRL-68. Apoptosis was further confirmed and quantified by DNA fragmentation ELISA, Annexin V (AnV) binding of externalized phosphatidylserine and the mitochondrial probe JC-1 using flow cytometry. The rapid increase in intracellular reactive oxygen species (ROS) levels was involved in the mechanism of cell death. Western blot analysis demonstrated that delta-elemene activated the caspase-signaling pathway, leading to the proteolysis conversion of pro-caspase-3 to activate caspase-3, and the subsequent cleavage of the caspase substrate PARP. In the process of the induction of apoptotic cell death, Bax translocated into mitochondria, a reduction in Deltapsim was observed and a release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria into the cytosol occurred, indicating that cell death induced by delta-elemene was through a mitochondrial-mediated pathway.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/patologia , Mitocôndrias/fisiologia , Sesquiterpenos/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Curcuma , Relação Dose-Resposta a Droga , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
This study was designed to investigate the apoptosis-inducing activity of delta-elemene on Hela cells in vitro. MTT assay and Hoechst 33258/PI fluorescence microscopy were used for this investigation. Apoptosis was further confirmed and quantified by DNA fragmentation ELISA, Annexin V (AnV) binding of externalized phosphatidylserine and the mitochondrial probe JC-1 using flow cytometry. Generation of reactive oxygen species (ROS) was detected using CM-H2DCFDA. Western blots analysis was performed using antibodies against the pro-caspase-3, or PRAP (Poly (ADP-ribose) polymerase). The results showed that delta-elemene exhibited a marked antiproliferative effect on Hela cells in dose- and time-dependent manners, and had little inhibition to normal human liver cell line WRL-68. It was demonstrated that delta-elemene was capable of inducing DNA fragmentation in a dose- and time-dependent manner. AnV positivity and the disturbance of the polarized mitochondrial transmembrane potential (Deltapsim) suggested that delta-elemene induced apoptotic death of Hela cells. Western blot analysis demonstrated that delta-elemene activated the caspase-signaling pathway, leading to the proteolysis conversion of pro-caspase-3 to activate caspase-3, and the subsequent cleavage of the caspase substrate PARP. Further, it was noted that the apoptotic effect of delta-elemene could be attenuated by L-Glutathione (GSH) or z-DEVD-fmk. It suggested that the increase in ROS generation might be involved in the mechanism of delta-elemene induced cell apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Sesquiterpenos/farmacologia , Caspase 3/metabolismo , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutationa/farmacologia , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oligopeptídeos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sesquiterpenos/antagonistas & inibidores , Estimulação QuímicaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV.
Assuntos
Acetilglucosamina/análise , Amino Açúcares/análise , Vírus da Síndrome Respiratória e Reprodutiva Suína/química , Ácidos Siálicos/análise , Proteínas do Envelope Viral/química , Precipitação Química , Glicosídeo Hidrolases/metabolismo , Lectinas/metabolismo , Espectrometria de MassasRESUMO
SCOPE: This study examined the associations of serum transferrin and metabolic disorders among Chinese population. METHODS AND RESULTS: This study is based on nation-wide, population-based China Health and Nutrition survey including 8564 men and women aged 18 years or older. Anthropometric and fasting blood glucose, insulin, lipids, and transferrin data were collected. Elevated transferrin concentrations associated with higher body mass index, waist circumference, lipids, insulin, glucose (all p < 0.0001). Serum transferrin concentrations increased gradually with increasing numbers of metabolic syndrome (MetS) components among men and women (p = 0.0003). Elevated concentrations of transferrin were significantly related with higher risk of MetS (p = 0.0006), obesity (p = 0.0089), overweight (p < 0.0001). No associations between transferrin concentrations and risk of diabetes and high blood pressure were observed in both men and women. CONCLUSION: Elevated transferrin concentrations were positively associated with risk of MetS and obesity, but not with risk of diabetes among Chinese.
Assuntos
Síndrome Metabólica/sangue , Obesidade/sangue , Sobrepeso/sangue , Transferrina/análise , Regulação para Cima , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , China/epidemiologia , Estudos Transversais , Feminino , Humanos , Estudos Longitudinais , Masculino , Síndrome Metabólica/epidemiologia , Síndrome Metabólica/etnologia , Síndrome Metabólica/fisiopatologia , Pessoa de Meia-Idade , Inquéritos Nutricionais , Obesidade/epidemiologia , Obesidade/etnologia , Obesidade/fisiopatologia , Sobrepeso/epidemiologia , Sobrepeso/etnologia , Sobrepeso/fisiopatologia , Fatores de Risco , Índice de Gravidade de Doença , Circunferência da Cintura/etnologia , Adulto JovemRESUMO
Quantitation is an essential aspect of comprehensive glycomics study. Here, a novel isotopic-labeling method is described for N-glycan quantitation using (18)O-water. The incorporation of the (18)O-labeling into the reducing end of N-glycans is simply and efficiently achieved during peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidase F release. This process provides a 2-Da mass difference compared with the N-glycans released in (16)O-water. A mathematical calculation method was also developed to determine the (18)O/(16)O ratios from isotopic peaks. Application of this method to several standard glycoprotein mixtures and human serum demonstrated that this method can facilitate the relative quantitation of N-glycans over a linear dynamic range of two orders, with high accuracy and reproducibility.
Assuntos
Glicoproteínas/química , Marcação por Isótopo/métodos , Polissacarídeos/análise , Glicômica/métodos , Humanos , Espectrometria de Massas , Isótopos de Oxigênio , Polissacarídeos/sangue , Polissacarídeos/química , Reprodutibilidade dos TestesRESUMO
Oviduct-specific glycoprotein (OVGP1) is a major mucin-like glycoprotein synthesized and secreted exclusively by non-ciliated secretory cells of mammalian oviduct. In vitro functional studies showed that OVGP1 plays important roles during fertilization and early embryo development. We have recently produced recombinant human oviduct-specific glycoprotein (rhOVGP1) in human embryonic kidney 293 (HEK293) cells. The present study was undertaken to characterize the structures and determine the biosynthetic pathways of the N- and O-glycans of rhOVGP1. Treatment of the stable rhOVGP1-expressing HEK293 cells with either GalNAcα-Bn to block O-glycan extension, tunicamycin to block N-glycosylation, or neuraminidase increased the electrophoretic mobility of rhOVGP1. A detailed analysis of O- and N-linked glycans of rhOVGP1 by mass spectrometry showed a broad range of many simple and complex glycan structures. In order to identify the enzymes involved in the glycosylation of rhOVGP1, we assayed glycosyltransferase activities involved in the assembly of O- and N-glycans in HEK293 cells, and compared these to those from the immortalized human oviductal cells (OE-E6/E7). Our results demonstrate that HEK293 and OE-E6/E7 cells exhibit a similar spectrum of glycosyltransferase activities that can synthesize elongated and sialylated O-glycans with core 1 and 2 structures, as well as complex multiantennary N-glycans. It is anticipated that the knowledge gained from the present study will facilitate future studies of the role of the glycans of human OVGP1 in fertilization and early embryo development.
Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Polissacarídeos/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Linhagem Celular , Glicoproteínas/genética , Humanos , Estrutura Molecular , Polissacarídeos/química , Proteínas Recombinantes/genéticaRESUMO
Fourteen beta-elemene derivatives containing a piperazine, a morpholine, a tetrahydropyrrole, a thiophenylethylamine, or a cyclohexamine group were synthesized. The structures of these beta-elemene derivatives were characterized with IR, 1H NMR, MS, and elemental analyses. All these derivatives had an increased anti-proliferative activity in human cervix epitheloid carcinoma HeLa, gastric carcinoma SGC-7901, and leukemia K562 cells comparing with that of beta-elemene. Among these derivatives, 13,14-bis(cis-3,5-dimethyl-1-piperazinyl)-beta-elemene (IIi), 13,14-bis[2-(2-thiophenyl)ethylamino]-beta-elemene (IIm), and 13,14-bis(cyclohexamino)-beta-elemene (IIn) were the most potent agents. IIi, IIm, and IIn inhibited K562 cell growth with an IG50 below 5 microM that was correlated with mTOR activity inhibition.