Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 124
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Methods Mol Biol ; 2274: 127-138, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050468

RESUMO

Spectral overlaps in fluorescence (FL) and bioluminescence (BL) commonly cause optical cross talks. The present protocol introduces five different lineages of coelenterazine (CTZ) analogues, which have selectivity to a specific luciferase, and thus cross talk-free. For example, some CTZ analogues with ethynyl or styryl groups display dramatically biased BL to specific luciferases and pH by modifying the functional groups at the C-2 and C-6 positions of the imidazopyridinne backbone of CTZ. The optical cross talk-free feature is exemplified with the multiplex system, which simultaneously illuminated antiestrogenic and rapamycin activities without optical cross talks. This unique protocol contributes to specific and high-throughput BL imaging of multiple optical readouts in mammalian cells without optical contamination.


Assuntos
Alcinos/química , Imidazóis/química , Luciferases/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Óptica/métodos , Pirazinas/química , Estirenos/química , Animais , Células COS , Chlorocebus aethiops , Moduladores de Receptor Estrogênico/farmacologia , Imunossupressores/farmacologia , Luciferases/efeitos dos fármacos , Sirolimo/farmacologia
2.
J Biomed Nanotechnol ; 13(2): 221-31, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29377653

RESUMO

Biomedical applications for metal and metal oxide nanoparticles are rapidly increasing. Here their functional impact on two well-characterized model enzymes, Luciferase (Luc) or ß-galactosidase (ß-Gal) was quantitatively compared. Nickel oxide nanoparticle (NiO-NP) activated ß-Gal (>400% control) and boron carbide nanoparticle (B4C-NP) inhibited Luc(<10% control), whereas zinc oxide (ZnO-NP) and cobalt oxide (Co3O4-NP) activated ß-Gal to a lesser extent and magnesium oxide (MgO) moderately inhibited both enzymes. Melanoma specific killing was in the order; ZnO > B4C ≥ Cu > MgO > Co3O4 > Fe2O3 > NiO, ZnO-NP inhibiting B16F10 and A375 cells as well as ERK enzyme (>90%) and several other cancer-associated kinases (AKT, CREB, p70S6K). ZnO-NP or nanobelt (NB) serve as photoluminescence (PL) cell labels and inhibit 3-D multi-cellular tumor spheroid (MCTS) growth and were tested in a mouse melanoma model. These results demonstrate nanoparticle and enzyme specific biochemical activity and suggest their utility as new tools to explore the important model metastatic foci 3-D environment and their chemotherapeutic potential.


Assuntos
Antineoplásicos/farmacologia , Melanoma Experimental/metabolismo , Nanopartículas Metálicas/química , Esferoides Celulares/efeitos dos fármacos , Óxido de Zinco/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Luciferases/análise , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , Metais Pesados/farmacologia , Camundongos , Óxido de Zinco/química , beta-Galactosidase/análise , beta-Galactosidase/efeitos dos fármacos , beta-Galactosidase/metabolismo
3.
Toxicol Appl Pharmacol ; 304: 9-17, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27211842

RESUMO

The exponential increase in the total number of engineered nanoparticles in consumer products requires novel tools for rapid and cost-effective toxicology screening. In order to assess the oxidative damage induced by nanoparticles, toxicity test systems based on a human HSPA1A promoter-driven luciferase reporter in HepG2, LO2, A549, and HBE cells were established. After treated with heat shock and a group of silver nanoparticles (AgNPs) with different primary particle sizes, the cell viability, oxidative damage, and luciferase activity were determined. The time-dependent Ag(+) ions release from AgNPs in cell medium was also evaluated. Our results showed that heat shock produced a strong time-dependent induction of relative luciferase activity in the four luciferase reporter cells. Surprisingly, at 4h of recovery, the relative luciferase activity was >98× the control level in HepG2-luciferase cells. Exposure to different sizes of AgNPs resulted in activation of the HSPA1A promoter in a dose-dependent manner, even at low cytotoxic or non-cytotoxic doses. The smaller (5nm) AgNPs were more potent in luciferase induction than the larger (50 and 75nm) AgNPs. These results were generally in accordance with the oxidative damage indicated by malondialdehyde concentration, reactive oxygen species induction and glutathione depletion, and Ag(+) ions release in cell medium. Compared with the other three luciferase reporter cells, the luciferase signal in HepG2-luciferase cells is obviously more sensitive and stable. We conclude that the luciferase reporter cells, especially the HepG2-luciferase cells, could provide a valuable tool for rapid screening of the oxidative damage induced by AgNPs.


Assuntos
Proteínas de Choque Térmico HSP70/biossíntese , Nanopartículas Metálicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Prata/toxicidade , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Genes Reporter/efeitos dos fármacos , Glutationa/metabolismo , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Células Hep G2 , Humanos , Luciferases/efeitos dos fármacos , Malondialdeído/metabolismo , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Tamanho da Partícula , Regiões Promotoras Genéticas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade
4.
J Biosci Bioeng ; 122(4): 385-92, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27038670

RESUMO

In our previous study, we used a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, AmyI-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; AmyI-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that AmyI-1-18 strongly bound to RNA with a KD value of 1.4 × 10(-8) M but not to DNA and that AmyI-1-18 specifically bound to DnaK with a KD value of 4.4 × 10(-6) M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Oryza/enzimologia , Fragmentos de Peptídeos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , alfa-Amilases/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cátions/química , Cátions/farmacologia , Sistema Livre de Células , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Insetos/farmacologia , Luciferases/química , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , Fragmentos de Peptídeos/química , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , alfa-Amilases/farmacologia
5.
Chem Commun (Camb) ; 48(89): 11011-3, 2012 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-23037447

RESUMO

The effects of two C(60) derivatives: C(60)[C(COOH)(2)](3) and C(60)(NH(2))(x)(OH)(y), have been investigated on the bioluminescence of humanized Gaussia luciferase (hGluc) for the first time. Utilizing the dual properties of carboxyl C(60) with luminescence quenching and free radicals scavenging, a novel BRET system was constructed for protease detection with high sensitivity.


Assuntos
Fulerenos/química , Luciferases/análise , Fulerenos/farmacologia , Limite de Detecção , Luciferases/efeitos dos fármacos , Luminescência , Estrutura Molecular , Peptídeo Hidrolases/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
J Nat Prod ; 75(7): 1332-8, 2012 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-22799262

RESUMO

Four new terpenylated coumarins (1-4) were isolated from the stem bark of Ailanthus altissima by bioactivity-guided fractionation using an in vitro SIRT1 deacetylation assay. Their structures were identified as (2'R,3'R)-7-(2',3'-dihydroxy-3',7'-dimethylocta-6'-enyloxy)-6,8-dimethoxycoumarin (1), 6,8-dimethoxy-7-(3',7'-dimethylocta-2',6'-dienyloxy)coumarin (2), (2'R,3'R,6'R)-7-(2',3'-dihydroxy-6',7'-epoxy-3',7'-dimethyloctaoxy)-6,8-dimethoxycoumarin (3), and (2'R,3'R,4'S,5'S)-6,8-dimethoxy-7-(3',7'-dimethyl-4',5'-epoxy-2'-hydroxyocta-6'-enyloxy)coumarin (4). Compounds 1-4 strongly enhanced SIRT1 activity in an in vitro SIRT1-NAD/NADH assay and an in vivo SIRT1-p53 luciferase assay. These compounds also increased the NAD-to-NADH ratio in HEK293 cells. The present results suggest that terpenylated coumarins from A. altissima have a direct stimulatory effect on SIRT1 deacetylation activity and may serve as lead molecules for the treatment of some age-related disorders.


Assuntos
Ailanthus/química , Cumarínicos/isolamento & purificação , Sirtuína 1/efeitos dos fármacos , Sequência de Aminoácidos , Cumarínicos/química , Humanos , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , Estrutura Molecular , NAD/análise , NAD/metabolismo , Ressonância Magnética Nuclear Biomolecular , Casca de Planta/química , Caules de Planta/química , República da Coreia , Estereoisomerismo
7.
Environ Sci Technol ; 45(4): 1695-702, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21250674

RESUMO

The response of a murine macrophage cell line exposed to a library of seven metal and metal oxide nanoparticles was evaluated via High Throughput Screening (HTS) assay employing luciferase-reporters for ten independent toxicity-related signaling pathways. Similarities of toxicity response among the nanoparticles were identified via Self-Organizing Map (SOM) analysis. This analysis, applied to the HTS data, quantified the significance of the signaling pathway responses (SPRs) of the cell population exposed to nanomaterials relative to a population of untreated cells, using the Strictly Standardized Mean Difference (SSMD). Given the high dimensionality of the data and relatively small data set, the validity of the SOM clusters was established via a consensus clustering technique. Analysis of the SPR signatures revealed two cluster groups corresponding to (i) sublethal pro-inflammatory responses to Al2O3, Au, Ag, SiO2 nanoparticles possibly related to ROS generation, and (ii) lethal genotoxic responses due to exposure to ZnO and Pt nanoparticles at a concentration range of 25-100 µg/mL at 12 h exposure. In addition to identifying and visualizing clusters and quantifying similarity measures, the SOM approach can aid in developing predictive quantitative-structure relations; however, this would require significantly larger data sets generated from combinatorial libraries of engineered nanoparticles.


Assuntos
Nanopartículas Metálicas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , Macrófagos , Nanopartículas Metálicas/química , Camundongos , Nanoestruturas , Óxidos/química , Óxidos/toxicidade
8.
Environ Sci Technol ; 45(4): 1623-9, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21194196

RESUMO

The concept of hormesis has generated considerable interest within the environmental and toxicological communities over the past decades. However, toxicological evaluation and prediction of hormesis in mixtures are challenging and only just unfolding. The hormetic effects of ten ionic liquids (ILs), singly and in mixtures in the ratios of their individual EC50, EC10, EC0, and ECm (maximal stimulatory effect concentration), on luciferase luminescence were determined by using microplate toxicity analysis. There was good agreement between the effects observed and predicted by concentration addition (CA) for all four mixtures. This evidence supports the use of CA model as a default approach for assessing the combined effect of chemicals at the molecular level. Focusing on the selected points of the concentration-response curves (CRCs) of mixtures, the mixtures of IL chemicals mixed at concentrations that individually showed stimulatory effects could produce inhibitory or no effects, and the mixture of IL chemicals mixed at concentrations that individually showed no effects could produce significant inhibitory effect. The three interesting phenomena in mixture hormesis may have important implications for current risk assessment practices.


Assuntos
Poluentes Ambientais/toxicidade , Hormese , Líquidos Iônicos/toxicidade , Luciferases/efeitos dos fármacos , Previsões , Íons , Medição de Risco
9.
Zhongguo Zhong Yao Za Zhi ; 36(18): 2530-4, 2011 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-22256760

RESUMO

OBJECTIVE: To establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method. METHOD: Human ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed. RESULT: The recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity. CONCLUSION: A phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.


Assuntos
Cicer/química , Avaliação Pré-Clínica de Medicamentos/métodos , Luciferases/efeitos dos fármacos , Fitoestrógenos/análise , Fitoestrógenos/farmacologia , Extratos Vegetais/farmacologia , Linhagem Celular Tumoral , Cicer/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Genes Reporter/efeitos dos fármacos , Vetores Genéticos/metabolismo , Genisteína/química , Genisteína/farmacologia , Humanos , Luciferases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Plasmídeos/efeitos dos fármacos , Plasmídeos/metabolismo , Transfecção/métodos
10.
J Nat Prod ; 72(12): 2163-8, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19928832

RESUMO

From the heartwood of Dalbergia parviflora, eight new compounds, khrinones A (1), B (2), C (3), D (4), and E (5), isodarparvinol B (6), dalparvin (7), and (3S)-sativanone (22), along with 32 known compounds, have been isolated and characterized as 17 isoflavones, nine isoflavanones, five flavanones, six isoflavans, and three miscellaneous substances. Isolates were evaluated for their cell proliferation stimulatory activity against the MCF-7 and T47D human breast cancer cell lines, and their luciferase inductive effects using luciferase transiently transfected MCF-7/luc and T47D/luc cells were also determined. Isoflavones such as genistein (10), biochanin A (11), tectorigenin (12), and 2'-methoxyformononetin (13) stimulated the proliferation of both cells, and concentrations of lower than 1 muM of these compounds showed equivalent activity to 10 pM of estradiol (E2). The new isoflavanone (22) also showed activity against both cell types, although it was weaker than that of the corresponding isoflavone (2'-methoxyformononetin, 13). Two optically active isoflavanones (22 and 24: (3S)-violanone) stimulated the proliferation of both cell lines at lower concentrations than three racemates (21: vestitone, 23: 7,3'-dihydroxy-4'-methoxyisoflavanone, and 25: 3-O-methylviolanone). Bowdichione (20), an isoflavone with a quinone structure in its B-ring, showed activity against only one cell line associated with MCF-7 in these assays.


Assuntos
Dalbergia/química , Estrogênios/isolamento & purificação , Estrogênios/farmacologia , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Plantas Medicinais/química , Ensaios de Seleção de Medicamentos Antitumorais , Estrogênios/química , Feminino , Flavonoides/química , Humanos , Luciferases/efeitos dos fármacos , Estrutura Molecular , Estereoisomerismo , Tailândia , Madeira/química
11.
Cancer Res ; 69(10): 4407-14, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19435895

RESUMO

Several mucosotropic human papillomaviruses (HPV), including HPV type 16 (HPV-16), are etiologic agents of a subset of anogenital cancers and head and neck squamous cell carcinomas. In mice, HPV-16 E7 is the most potent of the papillomaviral oncogenes in the development of cervical disease. Furthermore, interfering specifically with the expression of E7 in HPV-positive cell lines derived from human cervical cancers inhibits their ability to proliferate, indicating that the expression of E7 is important in maintaining the transformed phenotype in vitro. To assess the temporal role of E7 in maintaining HPV-associated tumors and precancerous lesions in vivo, we generated Bi-L E7 transgenic mice that harbor a tetracycline-inducible transgene that expresses both HPV-16 E7 and firefly luciferase. When we crossed Bi-L E7 mice to a K5-tTA transgene-inducing line of mice, which expresses a tetracycline-responsive transactivator selectively in the stratified squamous epithelia, the resulting Bi-L E7/K5-tTA bitransgenic mice expressed E7 and luciferase in the skin and cervical epithelium, and doxycycline repressed this expression. Bitransgenic mice displayed several overt and acute epithelial phenotypes previously shown to be associated with the expression of E7, and these phenotypes were reversed on treatment with doxycycline. Repressing the expression of E7 caused the regression of high-grade cervical dysplasia and established cervical tumors, indicating that they depend on the continuous expression of E7 for their persistence. These results suggest that E7 is a relevant target not only for anticancer therapy but also for the treatment of HPV-positive dysplastic cervical lesions.


Assuntos
Papillomavirus Humano 16 , Proteínas Oncogênicas Virais/genética , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Colo do Útero/efeitos dos fármacos , Colo do Útero/patologia , Primers do DNA , Replicação do DNA/efeitos dos fármacos , Doxiciclina/farmacologia , Feminino , Vaga-Lumes/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica , Genes Reporter , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Luciferases/efeitos dos fármacos , Luciferases/genética , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/efeitos dos fármacos , Proteínas E7 de Papillomavirus , Reação em Cadeia da Polimerase
12.
J Pharmacol Sci ; 109(3): 380-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19270430

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) and dioxins are ubiquitous environmental pollutants and activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. It has been reported that testosterone represses 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced transcription of the cytochrome P450 (CYP) 1A1 gene in LNCaP cells. In this study, we investigated the mechanism for the repression of 3-methylcholanthrene (3MC)-induced transcription of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, and AhR repressor (AhRR), by 5alpha-dihydroteststerone (DHT) in LNCaP and T47D cells, which are androgen receptor (AR)- and AhR-positive. Real-time PCR analysis showed that DHT repressed 3MC-induced mRNA expression of the CYP1 family and AhRR genes. DHT repressed 3MC-induced luciferase activity in an AhR response element-driven luciferase reporter assay in LNCaP and T47D cells. The inhibitory effect of DHT was abolished by knockdown of AR protein with siRNA. The protein levels of AhR and AhR nuclear translocator (Arnt), the AhR-dimerizing partner, were not affected by DHT. Co-immunoprecipitation assay showed that DHT significantly facilitated the complex formation between AR and AhR in 3MC-treated cells. These results suggest that complex formation between activated AR and AhR plays an important role in the suppression of 3MC-induced transcription of CYP1 family genes by DHT.


Assuntos
Androgênios/farmacologia , Di-Hidrotestosterona/farmacologia , Metilcolantreno/toxicidade , Transcrição Gênica/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/efeitos dos fármacos , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1B1 , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoprecipitação , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , Masculino , Reação em Cadeia da Polimerase , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/genética
13.
Toxicol In Vitro ; 22(8): 1935-47, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18835349

RESUMO

We have constructed two vector systems (pDMS5, pSAB2) containing the promoter regions of the human CYP1A1 gene including xenobiotic response elements or the promoter region of the Xenopus laevis vitellogenin A2 gene including estrogen response elements, respectively, and the genes for green fluorescent protein and firefly luciferase. These vectors were transfected into CHO-K1 cells. Transiently transfected cells consistently responded to 1 nmol/l TCDD (dioxin) or 10 nmol/l 17ss-estradiol, respectively, with a 3-5 fold increase in luciferase activity. Permanent cell lines were selected by culturing transiently transfected cells under continued presence of antibiotics and dilution cloning. Cells which had stably integrated the vector-DNA into the genomic DNA were selected. SiF6 cells responded to treatment with TCDD, PCB126, benzo(a)pyrene or indirubin-3'-monoxime in the concentration range between 0 and 1 micromol/l. SiG12 cells responded to treatment with bisphenol A, 4-MBC and DDT in the concentration range between 0 and 10 micromol/l. Compared with the controls, luciferase mRNA-abundance (semi-quantitative RT-PCR) and luciferase activity (luminescence assay) were elevated up to 3-fold. Resveratrol or tamoxifen, respectively, worked as full antagonists. Luciferase expression was increased upon treatment of cells with extracts of spiked soil samples indicating that our systems are suited for screening of environmental samples.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Poluentes Ambientais/toxicidade , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Citocromo P-450 CYP1A1/genética , Poluentes Ambientais/análise , Expressão Gênica , Genes Reporter/genética , Vetores Genéticos/metabolismo , Humanos , Luciferases/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Estrogênio/metabolismo , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção/métodos , Vitelogeninas/genética , Xenobióticos/análise , Xenobióticos/toxicidade , Xenopus laevis
14.
Biochem Biophys Res Commun ; 372(2): 341-5, 2008 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-18501191

RESUMO

Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd2+, Hg2+ and Pb2+ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC(50) in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far.


Assuntos
Cádmio/toxicidade , Chumbo/toxicidade , Mercúrio/toxicidade , Metais Pesados/toxicidade , Dobramento de Proteína , Adenosina Trifosfatases/efeitos dos fármacos , Cátions Bivalentes/farmacologia , Luciferases/química , Luciferases/efeitos dos fármacos , Chaperonas Moleculares/efeitos dos fármacos , Renaturação Proteica/efeitos dos fármacos
15.
Toxicol Appl Pharmacol ; 229(3): 386-98, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18336852

RESUMO

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-alpha (TNF-alpha) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-alpha in human A549 cells remain unclear. Here, we showed that TNF-alpha induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-kappaB (helenalin), and transfection with dominant negative mutants of ERK2 (DeltaERK) and JNK (DeltaJNK), and siRNAs for MEK1, p42 and JNK2. TNF-alpha-stimulated phosphorylation of p42/p44 MAPK and JNK were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of DeltaERK and DeltaJNK. Furthermore, the involvement of NF-kappaB in TNF-alpha-induced MMP-9 production was consistent with that TNF-alpha-stimulated degradation of IkappaB-alpha and translocation of NF-kappaB into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-kappaB was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-alpha in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-alpha-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-kappaB MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-alpha-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-kappaB are essential for TNF-alpha-induced MMP-9 gene expression.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos
16.
Pharmacology ; 81(4): 317-24, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18322419

RESUMO

The antineoplastic drug paclitaxel is known to block cells in the G2/M phase of the cell cycle through stabilization of microtubules. The development of paclitaxel resistance in tumors is one of the most significant obstacles to successful therapy. Vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) are important regulators of neovascularization. HIF-1 regulates VEGF expression at the transcriptional level. Here, we investigated whether paclitaxel treatment affects VEGF expression for the development of paclitaxel resistance. Paclitaxel treatment induced dose-dependent cell death and increased VEGF expression. Paclitaxel also induced nuclear factor-kappaB activation and stabilized HIF-1alpha, which stimulated luciferase activity of HIF-1alpha response element on VEGF gene. As paclitaxel treatment produced reactive oxygen species (ROS), VEGF expression was increased by H2O2 treatment and reduced by various ROS scavengers such as N-acetyl-L-cysteine, pyrrolidine dithiocarbamate and diphenylene iodonium. Paclitaxel-induced cell death was aggravated by incubation with those ROS scavengers. Collectively, this suggests that paclitaxel-induced VEGF expression could be mediated by paclitaxel-induced ROS production through nuclear factor-kappaB activation and HIF-1alpha stabilization, which could affect resistance induction to antitumor therapeutics during cancer treatment.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Paclitaxel/farmacologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Antineoplásicos Fitogênicos/administração & dosagem , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Paclitaxel/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
Int J Pharm ; 354(1-2): 196-203, 2008 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-18155371

RESUMO

Three cationic cholesterol derivatives (CCDs), which differ in their types of amine and bear a hydroxyethyl group at the amine group, were synthesized and formulated into liposomes and nanoparticles as gene delivery vectors. In vitro transfection into A549 cells proved that liposomes formulated with CCDs and dioleoylphosphatidylethanolamine (DOPE) of 1/2 molar ratio were more effective than the corresponding nanoparticles with CCDs and Tween 80 at charge ratios (+/-) of 1/2, 3/1 and 5/1. Among the liposomal formulations, non-hydroxyethylated CCDs were more effective than hydroxyethylated ones in vitro. However, gene transfection in the lung through intratracheal injection showed opposite results to those in vitro, with liposomes containing hydroxyethylated CCDs being more potent than those containing non-hydroxyethylated CCDs. Transfection by liposomes with N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (MHAPC) showed the highest luciferase activity, resulting in 2- and 60-fold higher gene expression than jet-PEI and naked DNA, respectively. The distribution of MHAPC lipoplex after intratracheal injection was heterogeneous, and luciferase was expressed in epithelial cells lining the bronchi and bronchioles. All the lipoplexes led to higher TNF-alpha levels in the lung compared to the nanoplex and jet-PEI, but our findings suggested that modification of the cationic cholesterol with a hydroxyethyl group at the tertiary amine terminal, MHAPC, promoted gene expression in the lung without increasing the toxicity compared with other CCDs. This work firstly proved that liposomes containing hydroxyethylated CCDs could promote gene expression in the lung through intratracheal injection.


Assuntos
Colesterol/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/química , Nanopartículas/administração & dosagem , Adenocarcinoma/genética , Animais , Cátions/química , Linhagem Celular Tumoral , Vetores Genéticos/administração & dosagem , Humanos , Lipossomos , Luciferases/efeitos dos fármacos , Luciferases/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Fosfatidiletanolaminas/química , Relação Estrutura-Atividade , Distribuição Tecidual , Transfecção/métodos
18.
Mol Imaging ; 7(6): 283-92, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19123998

RESUMO

Estrogen receptors have long been known to be expressed in several brain areas in addition to those directly involved in the control of reproductive functions. Investigations in humans and in animal models suggest a strong influence of estrogens on limbic and motor functions, yet the complexity and heterogeneity of neural tissue have limited our approaches to the full understanding of estrogen activity in the central nervous system. The aim of this study was to examine the transcriptional activity of estrogen receptors in the brain of male and female mice. Exploiting the ERE-Luc reporter mouse, we set up a novel, bioluminescence-based technique to study brain estrogen receptor transcriptional activity. Here we show, for the first time, that estrogen receptors are similarly active in male and female brains and that the estrous cycle affects estrogen receptor activity in regions of the central nervous system not known to be associated with reproductive functions. Because of its reproducibility and sensitivity, this novel bioluminescence application stands as a candidate as an innovative methodology for the study and development of drugs targeting brain estrogen receptors.


Assuntos
Encéfalo/metabolismo , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , Estro/fisiologia , Luciferases/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Diestro/fisiologia , Estradiol/farmacologia , Feminino , Fulvestranto , Luciferases/efeitos dos fármacos , Luminescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Proestro/fisiologia
19.
J Antibiot (Tokyo) ; 60(9): 547-53, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17917237

RESUMO

In the course of screening for an inhibitor of ER stress-induced XBP1 activation, we isolated a new member of the triene-ansamycin group compound, trierixin, from a culture broth of Streptomyces sp. AC 654. Trierixin was purified by column chromatography on silica gel and by HPLC. The molecular formula of trierixin is C(37)H(52)N(2)O(8)S. Trierixin inhibited thapsigargin-induced XBP1-luciferase activation in HeLa/XBP1-luc cells and endogenous XBP1 splicing in HeLa cells with an IC(50) of 14 ng/ml and 19 ng/ml, respectively. Moreover, in the process of isolating trierixin, we isolated structurally related mycotrienin II and trienomycin A as inhibitors of ER stress-induced XBP1 activation from a culture broth of a trierixin-producing strain. This study provides the first observation that triene-ansamycins have a novel inhibitory effect against XBP1 activation.


Assuntos
Antibacterianos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Streptomyces/química , Alanina/análogos & derivados , Alanina/isolamento & purificação , Antibacterianos/classificação , Antibacterianos/isolamento & purificação , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Fermentação , Células HeLa , Humanos , Hidroquinonas/isolamento & purificação , Concentração Inibidora 50 , Lactamas Macrocíclicas/classificação , Lactamas Macrocíclicas/isolamento & purificação , Luciferases/efeitos dos fármacos , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico/induzido quimicamente , Estresse Fisiológico/fisiopatologia , Tapsigargina , Fatores de Transcrição , Proteína 1 de Ligação a X-Box
20.
Immunobiology ; 212(8): 637-45, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17869641

RESUMO

B-cell activating factor (BAFF) plays a critical role for mature B cell generation and maintenance. We have previously described that mouse BAFF (mBAFF) transcript expression was increased by toll-like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS), through reactive oxygen species (ROS) production and NF-kappaB activation. Here, we investigated whether mBAFF expression could be regulated by promoter activation through the cooperation of NF-kappaB and p300, co-activator to various transcription factors. We cloned mBAFF promoter into luciferase-expressing pGL3-basic vector and computer-analyzed its NF-kappaB binding motif. Due to the existence of NF-kappaB binding motifs, activity in 2.0 kb mBAFF promoter was higher than that in 1.0 or 0.5 kb mBAFF promoter. When Raw 264.7 murine macrophages were stimulated with LPS, 2.0 kb mBAFF promoter activity was increased time dependently. Serum deprivation (0.5% FBS) producing ROS and exogenous H(2)O(2) treatment also enhanced mBAFF promoter activity, which was reduced by N-acetyl-l-cysteine (NAC), a well-known ROS scavenger. LPS and serum-starved ROS production increased NF-kappaB activation. mBAFF promoter activity was augmented by co-transfection with p65 and/or co-activator, p300. It was inhibited by dominant negative (DN) p300. Binding of p300 to BAFF promoter was detected by chromatin immunoprecipitation (ChIP) assay. Data suggest that mBAFF expression could be regulated by promoter activation through NF-kappaB activation, which might be dependent on the cooperation with co-activator, p300.


Assuntos
Fator Ativador de Células B/metabolismo , Proteína p300 Associada a E1A/metabolismo , Fator de Transcrição RelA/metabolismo , Acetilcisteína/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Peróxido de Hidrogênio/metabolismo , Luciferases/efeitos dos fármacos , Macrófagos , Camundongos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcrição Gênica , Ativação Transcricional , Transfecção
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...