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1.
Braz J Med Biol Res ; 53(5): e8457, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348428

RESUMO

The objective of this study was to investigate whether the conjugation of gold nanoparticles (GNPs) to 5-aminolevulinic acid (5-ALA) could enhance the anti-tumor efficiency of photodynamic therapy (PDT) in epidermoid carcinoma cells. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blot, respectively. Cell viability, apoptosis, invasion, and migration were determined by MTT assay, flow cytometry, transwell invasion assay, and migration assay, respectively. Singlet oxygen generation was detected by the singlet oxygen sensor green reagent assay. Our results showed that PDT with 5-ALA and GNPs-conjugated 5-ALA (5-ALA-GNPs) significantly suppressed cell viability, increased cell apoptosis and singlet oxygen generation in both HaCat and A431 cells, and PDT with 5-ALA and 5-ALA-GNPs had more profound effects in A431 cells than that in HaCat cells. More importantly, 5-ALA-GNPs treatment potentiated the effects of PDT on cell viability, cell apoptosis, and singlet oxygen generation in A431 cells compared to 5-ALA treatment. Further in vitro assays showed that PDT with 5-ALA-GNPs significantly decreased expression of STAT3 and Bcl-2 and increased expression of Bax in A431 cells compared with PDT with 5-ALA. In addition, 5-ALA-GNPs treatment enhanced the inhibitory effects of PDT on cell invasion and migration and Wnt/ß-catenin signaling activities in A431 cells compared to 5-ALA treatment. In conclusion, our results suggested that GNPs conjugated to 5-ALA significantly enhanced the anti-tumor efficacy of PDT in A431 cells, which may represent a better strategy to improve the outcomes of patients with cutaneous squamous cell carcinoma.

2.
Plant Biotechnol J ; 18(3): 756-769, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31469486

RESUMO

Nonspecific lipid transfer proteins (nsLTPs) play critical roles in plant development and response to abiotic stresses. Here, we found that a rice lipid transfer protein, OsLTPL159, was associated with cold tolerance at the early seedling stage. Overexpression of an OsLTPL159IL 112 allele from the cold-tolerant introgression line IL112 in either the japonica variety Zhonghua17 (ZH17) or the indica variety Teqing background dramatically enhanced cold tolerance. In addition, down-regulation of the expression of OsLTPL159 in the japonica variety ZH17 by RNA interference (RNAi) significantly decreased cold tolerance. Further transcriptomic, physiological and histological analysis showed that the OsLTPL159IL 112 allele likely enhanced the cold tolerance of rice at the early seedling stage by decreasing the toxic effect of reactive oxygen species, enhancing cellulose deposition in the cell wall and promoting osmolyte accumulation, thereby maintaining the integrity of the chloroplasts. Notably, overexpression of another allele, OsLTPL159GC 2 , from the recipient parent Guichao 2 (GC2), an indica variety, did not improve cold tolerance, indicating that the variations in the OsLTPL159 coding region of GC2 might disrupt its function for cold tolerance. Further sequence comparison found that all 22 japonica varieties surveyed had an OsLTPL159 haplotype identical to IL112 and were more cold-tolerant than the surveyed indica varieties, implying that the variations in OsLTPL159 might be associated with differential cold tolerance of japonica and indica rice. Therefore, our findings suggest that the OsLTPL159 allele of japonica rice could be used to improve cold tolerance of indica rice through a molecular breeding strategy.

4.
Cancer Chemother Pharmacol ; 84(4): 719-728, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31281953

RESUMO

PURPOSE: Glioblastoma is the most common, malignant and devastating type of primary brain tumor. Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is characterized by its lethality to precancerous and cancerous cells. However, many kinds of tumor cells, including most glioma cells, tend to evade TRAIL-induced apoptosis. Celastrol is a pleiotropic compound from a traditional Chinese medicine that has proven to be useful as a sensitizer for TRAIL treatment. However, the underlying mechanism and role of celastrol in the sensitization of glioma cells remain to be elucidated. METHODS: The viability of glioma cell lines was examined by the CCK-8 assay. The expression of DR5 was detected by reverse transcriptase quantitative real-time PCR. The protein expression of DR5, cleaved caspase-8, cleaved caspase-3 and PARP were measured by western blot. The apoptosis rates and the sub-G1 population were detected by flow cytometry. The cellular morphological changes were assessed by TUNEL apoptosis and Hoechst 33258 staining assays. The knockdown of DR5 expression was conducted by siRNA. RESULTS: In this study, we observed that celastrol treatment inhibited cell viability in a dose-dependent manner, while glioma and normal human astroglial cell lines were resistant to TRAIL treatment. We also observed that the antiproliferative effects of TRAIL in combination with a noncytotoxic concentration of celastrol were significantly greater than those of celastrol or TRAIL alone. In addition, cell death induced by the combination treatment was apoptotic and occurred through the death receptor pathway via activation of caspase-8, caspase-3, and PARP. Furthermore, celastrol upregulated death receptor 5 (DR5) at the mRNA and protein levels, and siRNA-mediated DR5 knockdown reduced the killing effect of the combination drug treatment on glioma cells and reduced the activation of caspase-3, caspase-8 and PARP. CONCLUSIONS: Taken together, the results of our study demonstrate that celastrol sensitizes glioma cells to TRAIL via the death receptor pathway and that DR5 plays an important role in the effects of this cotreatment. The results indicate that this cotreatment is a promising tumor-killing therapeutic strategy with high efficacy and low toxicity.

6.
J Cell Mol Med ; 23(5): 3097-3107, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30825266

RESUMO

BACKGROUND: Complement C3 has been shown to be highly expressed in cutaneous squamous cell carcinoma (cSCC) tumour tissues and is correlated with tumour cell growth. This study aimed to investigate the mechanism of C3 in cSCC malignant transformation. METHODS: C3 expression was analysed in cSCC cell lines A431, Tca8113, SCC13, HSC-5 and HSC-1 and in immortalized HaCaT keratinocytes. Proliferation and migration of cSCC were determined after C3a exposure. Expression of cyclin D1, cyclin E, vascular endothelial growth factor (VEGF), pro-matrix metalloproteinase 1 (pro-MMP1), pro-matrix metalloproteinase 2 (pro-MMP2), stemness factors, GSK-3ß, and ß-catenin were analyzed. Tumour growth was examined in a murine xenograft model. RESULTS: C3 expression was much more highly expressed in all cSCC cell lines than in HaCaT cells. C3a treatment significantly promoted cSCC cell proliferation and migration and upregulated cyclin D1, cyclin E, VEGF, pro-MMP1 and pro-MMP2 expression, which were impeded by the C3aR antagonist. Moreover, the expression of stemness factors Sox-2, Nanog, Oct-4, c-Myc and CD-44 was stimulated by C3a and slowed by C3aR disruption. Knockdown of Sox-2 by siRNA transfection suppressed cell proliferation and migration, constrained VEGF secretion and inhibited pro-MMP1 and pro-MMP2 expression. C3a also activated the Wnt and ß-catenin pathway in cSCC cells. Disruption of C3aR expression dampened tumour growth and the expression of Wnt-1, ß-catenin and Sox-2 in the xenograft model. CONCLUSIONS: C3a enhanced cell proliferation, migration and stemness in cSCC, and this activity was correlated with activation of the Wnt and ß-catenin pathway.

7.
Appl Opt ; 58(6): 1522-1529, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30874042

RESUMO

The large normal dispersion of the fundamental mode (TEn=1 mode) in the whispering gallery modes (WGM) microsphere is detrimental to the visible comb generation. Herein, we demonstrate that this fundamental limitation can be removed by considering the high-order radial modes (TEn=2 mode) of the hybrid microsphere cavity (HMC). The studied HMC consists of a high-refractive-index coating (TiO2 or HfO2) and silica microsphere. The simulated electric field energy distribution and measured Q value in our experiment show that optical confinement of the coating effectively excites the TEn=2 mode and reduces the free spectral range (FSR) and modal dispersion. In addition, the observed redshift of WGM and decreased trend of FSR are in accordance with simulations. The zero-dispersion wavelength can be linearly shifted to a shorter wavelength or even into the visible region with the reduction of coating thickness or refractive index and larger microcavity, which advances the visible comb generation.

8.
Environ Sci Pollut Res Int ; 25(22): 21623-21634, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29785600

RESUMO

High concentrations of grease easily inhibit anaerobic digestion. The stability of the process and microbial responses in the controlling internal circulation (CIC) reactor used for treating food waste were investigated under different grease contents and inner circulation ratios. Results showed that at the grease content of 1 g/L, the removal rates of 94% and 86-93% were achieved for chemical oxygen demand (COD) and NH3-N, respectively. In contrast, when the grease content increased to 7 g/L, removal rates for COD and NH3-N significantly decreased to 42.8 and 10%, respectively. In the three-dimensional excitation and emission matrix (3D-EEM) spectra of LB-EPS (loosely bound extracellular polymeric substances), the fluorescence intensity of coenzyme F420 was weakened in the granular sludge, and the fluorescence peak of aromatic protein disappeared in the TB-EPS (tightly bound EPS). The activity and stability of the granular sludge deteriorated with increasing grease content, in this case at 7 g/L. However, when the inner cycle ratio was increased to 4, the removal rate of COD and NH3-N increased to about 70 and 76%, respectively. The adverse effects of grease could be decreased by increasing the inner cycle ratio. When the grease content increased from 1 to 7 g/L, the abundance of Methanofollis increased from 9.93 to 46.41%, while Methanothrix abundance was reduced from 18.4 to 3.07%. It could indicate that Methanothrix was sensitive to high grease content.


Assuntos
Reatores Biológicos/microbiologia , Matriz Extracelular de Substâncias Poliméricas/química , Resíduos de Alimentos , Hidrocarbonetos , Microbiota , Análise da Demanda Biológica de Oxigênio , Nitrogênio/isolamento & purificação , Esgotos/química
9.
Tree Physiol ; 38(1): 66-82, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29036367

RESUMO

To investigate the physiological responses of poplars to amino acids as sole nitrogen (N) sources, Populus × canescens (Ait.) Smith plants were supplied with one of three nitrogen fertilizers (NH4NO3, phenylalanine (Phe) or the mixture of NH4NO3 and Phe) in sand culture. A larger root system, and decreased leaf size and CO2 assimilation rate was observed in Phe- versus NH4NO3-treated poplars. Consistently, a greater root biomass and a decreased shoot growth were detected in Phe-supplied poplars. Decreased enzymatic activities of nitrate reductase (NR), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) and elevated activities of nitrite reductase (NiR), phenylalanine ammonia lyase (PAL), glutamine synthetase (GS) and asparagine synthase (AS) were found in Phe-treated roots. Accordingly, reduced concentrations of NH4+, NO3- and total N, and enhanced N-use efficiencies (NUEs) were detected in Phe-supplied poplars. Moreover, the transcript levels of putative Phe transporters ANT1 and ANT3 were upregulated, and the mRNA levels of NR, glutamine synthetase 2 (GS2), NADH-dependent glutamate synthase (NADH-GOGAT), GDH and asparagine synthetase 2 (ASN2) were downexpressed in Phe-treated roots and/or leaves. The 15N-labeled Phe was mainly allocated in the roots and only a small amount of 15N-Phe was translocated to poplar aerial parts. These results indicate that poplar roots can acquire Phe as an N source to support plant growth and that Phe-induced NUEs in the poplars are probably associated with NH4+ re-utilization after Phe deamination and the carbon bonus simultaneously obtained during Phe uptake.


Assuntos
Nitrogênio/metabolismo , Fenilalanina/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Populus/metabolismo , Glutamato-Amônia Ligase/metabolismo , Nitrato Redutase/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Folhas de Planta/metabolismo
10.
Appl Opt ; 56(27): 7572-7576, 2017 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-29047733

RESUMO

We report an experimental realization of five-order Stokes stimulated Raman scattering lasing in silica microspheres pumped by a 1030 nm continuous-wave laser. The wavelength of the Stokes Raman laser is extended to 1348.55 nm, which is located in the second low loss window of the optical fiber. It has potential applications in the wavelength converter and Raman amplifier in O-waveband optical communication. The minimum pump power is about 50 µW when the first-order Stokes Raman laser can be observed.

11.
Huan Jing Ke Xue ; 37(10): 4062-4068, 2016 Oct 08.
Artigo em Chinês | MEDLINE | ID: mdl-29964444

RESUMO

With Chinese medicinal herbal residues and municipal sludge as raw materials for co-composting experiment, the effect of the material ratio and addition time of Chinese medicinal herbal residues on the composting efficiency were investigated, including the change of the temperature, organic matter, ammonia nitrogen, and activity of protease. The best composting conditions were determined based on the results. The experimental results showed that the temperature of the pile was raised in the presence of 60 g Chinese medicinal herbal residues as carbon source and 300 g municipal sludge, the ammonia volatilization was reduced and the activity of protease was improved. The ammonia volatilization was reduced by 35.9% and the activity of protease was increased by 80.5% in 15 d, respectively. Especially, in the early stage, addition of Chinese medicinal herbal residues as conditioner could increase the organic matter degradation. Thus, the composting process was accelerated. Changes in the UV-visible and fluorescence characteristics of dissolved organic matter (DOM) during the co-composting process were discussed. The treatment with Chinese medicinal herbal residues improved the maturity of the compost. Moreover, phospholipid fatty acid (PLFA) method was used to estimate the microbial community structure changes. It showed that the number of microbial community such as fungi and Gram negative bacteria increased with addition of Chinese medicinal herbal residues.


Assuntos
Amônia/química , Compostagem , Resíduos de Drogas/química , Medicamentos de Ervas Chinesas/química , Esgotos/química , Fungos , Bactérias Gram-Negativas , Solo
12.
Zhongguo Zhong Yao Za Zhi ; 41(3): 451-455, 2016 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-28868863

RESUMO

Rosa roxburghii, a kind of the medical and edible plants belonging to the Rosaceae family, is widely distributed in the southwest districts of China, especially Guizhou province. Now, by reason of the extensive bioactivities, the plant is widely used in the field of food, health product, drug, and so on. In the course of our continuing search for the bioactive constituents, thirteen compounds were isolated from R. roxburghii, and their structures were determined on the basis of physicochemical property, spectroscopic data and comparison with the literatures, as 2-oxo pomolic acid(1), 1ß-hydroxyeuscaphic acid(2), euscaphic acid(3), arjunic acid(4), tormentic acid(5), kaiiichigeside F1(6), rosamultin(7), arjunetin(8), 2ɑ, 3ɑ, 19ɑ-trihydroxy-olean-12-en-28-oic acid 28-O-ß-D-glucopyranoside(9), 2α, 3α, 19α, 24-tetrahydroxyolean-12-en-28-oic-acid 28-O-ß-D-glucopyranosyl ester(10), pyrogallic acid (11), daucosterol(12), and 1, 2-decanediol(13). Compounds 9 and 10 were firstly obtained from Rosaceae family, and compounds 1,4,5,9-11,13 were isolated from this plant for the first time.


Assuntos
Extratos Vegetais/química , Rosa/química , China , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Extratos Vegetais/isolamento & purificação , Plantas Comestíveis/química , Espectrometria de Massas por Ionização por Electrospray
13.
J Cell Mol Med ; 19(6): 1197-207, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25786906

RESUMO

Hirschsprung's disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. We investigated changes in expression of microRNAs (miRNAs) and the genes they regulate in tissues of patients with HSCR. Quantitative real-time PCR and immunoblot analyses were used to measure levels of miRNA, mRNAs, and proteins in colon tissues from 69 patients with HSCR and 49 individuals without HSCR (controls). Direct interactions between miRNAs and specific mRNAs were indentified in vitro, while the function role of miR-218-1 was investigated by using miR-218 transgenic mice. An increased level of miR-218-1 correlated with increased levels of SLIT2 and decreased levels of RET and PLAG1 mRNA and protein. The reductions in RET and PLAG1 by miR-218-1 reduced proliferation and migration of SH-SY5Y cells. Overexpression of the secreted form of SLIT2 inhibited cell migration via binding to its receptor ROBO1. Bowel tissues from miR-218-1 transgenic mice had nerve fibre hyperplasia and reduced numbers of gangliocytes, compared with wild-type mice. Altered miR-218-1 regulation of SLIT2, RET and PLAG1 might be involved in the pathogenesis of HSCR.


Assuntos
Proteínas de Ligação a DNA/genética , Doença de Hirschsprung/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-ret/genética , Receptores Imunológicos/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Colo/metabolismo , Colo/patologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Doença de Hirschsprung/metabolismo , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética
14.
J Cell Mol Med ; 18(8): 1580-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24974861

RESUMO

Hirschsprung's disease (HSCR), a congenital gastrointestinal disorder, is one of the most common causes of neonatal bowel obstruction. Without an early screening and diagnosis, some patients develop serious complications, such as toxic megacolon or acute enterocolitis. We sought to identify specific serum microRNAs (miRNAs) that can serve as novel early, non-invasive screening signature and then to test their specificity and sensitivity in diagnosing Hirschsprung's disease. We obtained serum samples from 95 HSCR cases and 104 matched controls. An initial screening of miRNA expression was performed through TaqMan Low Density Array. The candidate miRNAs were validated by individual reverse transcription quantitative real-time PCR arranged in the training and a two-stage validation set. Additional double-blind testing was performed in 23 patients with clinically suspected HSCR to evaluate the diagnostic value and accuracy of the serum miRNA profile in predicting HSCR. Following a multi-stage evaluation approach, five miRNAs were significantly increased in HSCR cases compared with controls. The areas under the receiver operating characteristic (ROC) curve of this five-serum miRNA signature were 0.895, 0.893 and 0.925 in training set and two validation sets, respectively. The accuracy rate of the five-miRNA profile as HSCR signature was 82.6%, which, in the double-blind testing set, was markedly higher than that of contrast enema (70%), the most commonly used test performed to diagnose HSCR. Our results indicate that a five-serum miRNA signature may be linked to HSCR, representing a potential, novel, non-invasive diagnostic approach for early screening of HSCR.


Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Doença de Hirschsprung/sangue , Doença de Hirschsprung/diagnóstico , MicroRNAs/genética , Estudos de Casos e Controles , Método Duplo-Cego , Feminino , Doença de Hirschsprung/genética , Humanos , Lactente , Masculino , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Cell Physiol Biochem ; 32(6): 1655-67, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24334875

RESUMO

BACKGROUND: MiR-141 has been confirmed to be associated with various human diseases. However, whether miR-141 is involved in the pathogenesis of Hirschsprung's disease (HSCR) remains unknown. Here, we design the experiment to reveal the relationship between miR-141 and HSCR. METHODS: Quantitative real-time PCR and Western blot were used to detect the expression levels of miR-141 and its potential genes in 70 tissues of HSCR compared with 60 controls. Bisulfite sequencing PCR (BSP) assay was applied to explain the possible mechanism of the aberrant expression level of miR-141. We employed a dual-luciferase reporter assay to validate the regulation relation between miR-141 and CD47/CUL3. Cell migration, proliferation, apoptosis, and cell cycle progression were examined by transwell assay, MTT assay, and flow cytometry, respectively. RESULTS: MiR-141 was down-regulated whereas CD47 and CUL3 expression was increased in colon tissues from patients with HSCR compared with control group, The increased level of CD47 and CUL3 induced by miR-141 reduced proliferation and migration of 293T and SH-SY5Y cells. Furthermore, this suppression was reversed by reducing of CD47 and CUL3. Hypermethylation of a CpG Island in the promoter region of miR-141 gene was confirmed in HSCR tissues. CONCLUSION: Aberrant reduction of miR-141 may play an important role in the pathogenesis of HSCR with the inhibiting affection on cell migration and proliferation abilities. The present study demonstrates for the first time the role of miR-141 and its target genes in the occurrence of HSCR, and provides us a new direction for the study of the pathogenesis of Hirschsprung's disease.


Assuntos
Proteínas Culina/metabolismo , Doença de Hirschsprung/patologia , MicroRNAs/metabolismo , Sequência de Bases , Antígeno CD47/química , Antígeno CD47/genética , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colo/metabolismo , Proteínas Culina/antagonistas & inibidores , Proteínas Culina/genética , Metilação de DNA , Regulação para Baixo , Feminino , Células HEK293 , Doença de Hirschsprung/metabolismo , Humanos , Lactente , Masculino , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Regulação para Cima
16.
J Pediatr Surg ; 48(10): 2099-105, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24094964

RESUMO

BACKGROUND/PURPOSE: Hirschsprung's disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells which are highly associated with impaired proliferation and migration of neural crest cells. Whether methyl CpG binding protein 2 (MeCP2) is related with HSCR still remains unknown. This study investigates the involvement of MeCP2 in HSCR. METHODS: Quantitative real time PCR and western blot were used to detect the expression level of MeCP2 both in the aganglionic/diseased segment and the ganglionic/normal segment. In vitro assays we used siRNAs to knock-down the expression of MeCP2 in SH-SY5Y cell lines, and furthermore, MTT and transwell assays were used to detect the proliferation and migration ability, respectively. In addition, bisulfite sequencing (BSP) and miRNA analysis were used to examine why MeCP2 is decreased in HSCR samples. RESULTS: MeCP2 exhibited a lower expression level in tissues of HSCR patients compared with the controls. The down-regulation may also suppress the proliferative ability of the cells. However, there was no significant difference in the MeCP2 methylation level between cases and controls. Similarly, there was no difference between cases and controls in miRNA-34b (miR-34b) which is predicted to regulate MeCP2 through complementary binding to the 3'-untranslated region of MeCP2. CONCLUSION: Our results indicated that an aberrant decreased level of MeCP2 may play an important role in the pathogenesis of HSCR.


Assuntos
Colo/metabolismo , Regulação para Baixo , Doença de Hirschsprung/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Reto/metabolismo , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Linhagem Celular , Movimento Celular , Proliferação de Células , Metilação de DNA , Feminino , Doença de Hirschsprung/genética , Humanos , Lactente , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
17.
Reprod Biol Endocrinol ; 11: 72, 2013 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-23889939

RESUMO

BACKGROUND: The ubiquitous use of dibutyl phthalate (DBP), one of the most widely used plasticizers, results in extensive exposure to humans and the environment. DBP and its major metabolite, monobutyl phthalate (MBP), may alter steroid biosynthesis and their exposure may lead to damage to male reproductive function. Low-doses of DBP/MBP may result in increased steroidogenesis in vitro and in vivo. However, the mechanisms of possible effects of low-dose MBP on steroidogenesis remain unclear. The aim of present study was to elaborate the role of transcription factors and steroidogenic acute regulatory protein in low-dose MBP-induced distruption of steroidogenesis in mouse Leydig tumor cells (MLTC-1 cells). METHODS: In the present study, MLTC-1 cells were cultured in RPMI 1640 medium supplemented with 2 g/L sodium bicarbonate. Progesterone level was examined by I125-pregesterone Coat-A-Count radioimmunoassay (RIA) kits. mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. DNA-binding of several transcription factors was examined by electrophoretic mobility shift assay (EMSA). RESULTS: In this study, various doses of MBP (0, 10(-9), 10(-8), 10(-7), or 10(-6) M) were added to the medium followed by stimulation of MLTC-1 cells with human chorionic gonadotrophin (hCG). The results showed that MBP increased progesterone production and steroidogenic acute regulatory protein (StAR) mRNA and protein levels. However, the protein levels of cytochrome P450scc and 3 beta-hydroxy-steroid dehydrogenase (3 beta-HSD) were unchanged after MBP treatment. EMSA assay showed that DNA-binding of steroidogenic factors 1(SF-1), GATA-4 and CCAAT/enhancer binding protein-beta (C/EBP-beta) was increased in a dose-dependent manner after MBP exposure. Western blot tests were next employed and confirmed that the protein levels of SF-1, GATA-4 and C/EBP-beta were also increased. Additionally, western blot tests confirmed the expression of DAX-1, negative factor of SF-1, was dose-dependently down regulated after MBP exposure, which further confirmed the role of SF-1 in MBP-stimulated steroid biosynthesis. CONCLUSIONS: In conclusion, we firstly delineated the regulation of StAR by transcription factors including SF-1, GATA-4 and C/EBP-beta maybe critical mechanism involved in low-dose MBP-stimulated steroidogenesis.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Fator de Transcrição GATA4/metabolismo , Fosfoproteínas/genética , Ácidos Ftálicos/farmacologia , Fator Esteroidogênico 1/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gonadotropina Coriônica/farmacologia , Receptor Nuclear Órfão DAX-1/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Tumor de Células de Leydig/genética , Tumor de Células de Leydig/metabolismo , Tumor de Células de Leydig/patologia , Masculino , Camundongos , Fosfoproteínas/metabolismo , Progesterona/biossíntese , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroides/biossíntese
18.
J Pediatr Surg ; 48(4): 813-7, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23583139

RESUMO

BACKGROUND/PURPOSE: Hirschsprung's disease (HSCR) is a common cause of neonatal bowel obstruction characterized by the absence of ganglion cells in the colon. Impaired migration of the neural crest cells (NCCs) has been implicated as one of the main causes of HSCR. E2F3, a member in the E2F family, which plays a crucial role in the control of the cell cycle is correlated with neuron migration. However, the function of E2F3 in the development of the enteric nervous system still remains unknown. This study aims to reveal the correlation of E2F3 in the progress of HSCR. METHODS: By using reverse transcriptase polymerase chain reaction (RT-PCR) and western blot assay, we investigated levels of E2F3 expression in 58 HSCR patients, both in the aganglionic bowel segment and the normal ganglionic segment, and in 39 unrelated controls. By in vitro assays, we used the siRNA method to knock-down the level of E2F3 expression in 293T cell lines. Furthermore, transwell assay was used to detect cell migration ability. RESULTS: Aberrant lower expression level of E2F3 was detected in the HSCR-S segment compared with the control group by RT-PCR and western blot assay. Besides, down-regulated E2F3 could suppress the cell migration. CONCLUSIONS: This is the first study showing the down-regulation of E2F3 in HSCR, bringing new insight to the mechanism of the impaired migration of neural crest cells.


Assuntos
Fator de Transcrição E2F3/metabolismo , Doença de Hirschsprung/metabolismo , Western Blotting , Linhagem Celular , Movimento Celular , Criança , Regulação para Baixo , Feminino , Doença de Hirschsprung/cirurgia , Humanos , Masculino , Crista Neural/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas
19.
Pediatr Surg Int ; 29(7): 683-8, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23579558

RESUMO

PURPOSE: Hirschsprung's disease (HSCR) is characterized by absence of the enteric nervous system in a variable portion of the distal gut. The endothelin receptor type B (EDNRB) gene has been localized to the chromosome 13q22 region and encodes a G-protein coupled receptor, is generally accepted as a crucial gene for HSCR. This study is to identify the epigenetic changes of EDNRB in the pathogenesis of HSCR. METHODS: We investigated the expression levels of EDNRB in 58 HSCR patients and 25 unrelated controls, using reverse transcriptase polymerase chain reaction (RT-PCR) and western blot assay. Moreover, using the methylation-specific polymerase chain reaction, we examined the methylation status of the promoter region of EDNRB. RESULTS: Aberrant high expression level of EDNRB was detected in HSCR patients compared with the control group (P = 0.023). Besides, western blot assay confirmed the up-regulation of EDNRB in the post transcription level in the aganglionosis segment of HSCR patients. Furthermore, there was a significantly lower ratio of methylation level of EDNRB in HSCR. CONCLUSIONS: Our study demonstrates that epigenetic inactivation of the EDNRB gene may play a role in the development of HSCR.


Assuntos
Colo/metabolismo , Doença de Hirschsprung/genética , Doença de Hirschsprung/metabolismo , Receptor de Endotelina B/genética , Western Blotting/métodos , China , Feminino , Humanos , Lactente , Masculino , Metilação , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
20.
Planta ; 237(4): 919-31, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23179443

RESUMO

Poplar plants are cultivated as woody crops, which are often fertilized by addition of ammonium (NH4(+)) and/or nitrate (NO3(-)) to improve yields. However, little is known about net NH4(+)/NO3(-) fluxes and their relation with H(+) fluxes in poplar roots. In this study, net NH4(+)/NO3(-) fluxes in association with H(+) fluxes were measured non-invasively using scanning ion-selective electrode technique in fine roots of Populus popularis. Spatial variability of NH4(+) and NO3(-) fluxes was found along root tips of P. popularis. The maximal net uptake of NH4(+) and NO3(-) occurred, respectively, at 10 and 15 mm from poplar root tips. Net NH4(+) uptake was induced by ca. 48 % with provision of NO3(-) together, but net NO3(-) uptake was inhibited by ca. 39 % with the presence of NH4(+) in poplar roots. Furthermore, inactivation of plasma membrane (PM) H(+)-ATPases by orthovanadate markedly inhibited net NH4(+)/NO3(-) uptake and even led to net NH4(+) release with NO3(-) co-provision. Linear correlations were observed between net NH4(+)/NO3(-) and H(+) fluxes in poplar roots except that no correlation was found between net NH4(+) and H(+) fluxes in roots exposed to NH4Cl and 0 mM vanadate. These results indicate that root tips play a key role in NH4(+)/NO3(-) uptake and that net NH4(+)/NO3(-) fluxes and the interaction of net fluxes of both ions are tightly associated with H(+) fluxes in poplar roots.


Assuntos
Compostos de Amônio/metabolismo , Nitratos/metabolismo , Raízes de Plantas/metabolismo , Populus/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Membrana Celular/enzimologia , Xilema/metabolismo
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