RESUMO
Surface properties are essential for substrates exhibiting high sensitivity in surface-enhanced Raman scattering (SERS) applications. In this work, novel SERS hybrid substrates using polystyrene-block-poly(methyl methacrylate) and anodic aluminum oxide templates is presented. The hybrid substrates not only possess hierarchical porous nanostructures but also exhibit superhydrophilic surface properties with the water contact angle ≈0°. Such surfaces play an important role in providing uniform enhanced intensities over large areas (relative standard deviation ≈10%); moreover, these substrates are found to be highly sensitive (limit of detection ≈10-12 m for rhodamine 6G (R6G)). The results show that the hybrid SERS substrates can achieve the simultaneous detection of multicomponent mixtures of different target molecules, such as R6G, crystal violet, and methylene blue. Furthermore, the bending experiments show that about 70% of the SERS intensities are maintained after bending from ≈30° to 150°.
Assuntos
Óxido de Alumínio/química , Polímeros/química , Rodaminas/análise , Molhabilidade , Eletrodos , Tamanho da Partícula , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
AIMS: Vinegar-baked Radix Bupleuri (VBRB) potentiates the activity of anticancer drugs in the liver by increasing their hepatic distribution. However, this phenomenon may be associated with drug transporters. We investigated the effect of saikosaponin b2 (SSb2; the main component of VBRB) on the activity and expression of different drug transporters in both normal cells and those that overexpress the transporter. MAIN METHODS: The activities of transporters were analyzed by concentration of their cellular substrates. Concentrations of colchicine (substrate of Pgp and MRP1) and cisplatin (substrate of OCT2 and MRP2) were determined by high-performance liquid chromatography (HPLC). The concentration of rhodamine B was determined by flow cytometry. The expression of transporter gene and protein were determined by qRT-PCR and Western blotting analysis. KEY FINDINGS: SSb2 increased colchicine efflux in HEK293 cells by primarily increasing Mrp1 activity, independent of gene and protein expression. SSb2 enhanced Mrp2 function and increased cisplatin efflux in BRL3A cells by upregulating Mrp2 gene expression, with a marginal effect on Pgp in normal cells. SSb2 increased OCT2 activity in OCT2-HEK293 cells by increasing the expression of OCT2 protein and mRNA; however, SSb2 inhibited MRP2 activity in MRP2-HEK293 cells by decreasing MRP2 protein expression, and decreased Pgp and MRP1 activity in Pgp- and MRP1-HEK293 cells. SIGNIFICANCE: SSb2 might potentially be the key active component of VBRB that enhances the hepatotargeting of anticancer drugs through the inhibition of multidrug resistance-associated drug transporters (Pgp, MRP1, and MRP2) in an environment-dependent manner.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Saponinas/metabolismo , Saponinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Cisplatino/análise , Cisplatino/metabolismo , Cisplatino/farmacologia , Colchicina/análise , Colchicina/metabolismo , Colchicina/farmacologia , Resistência a Múltiplos Medicamentos/fisiologia , Células HEK293 , Humanos , Medicina Tradicional Chinesa , Proteínas de Membrana Transportadoras/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacologia , RNA Mensageiro/metabolismo , Rodaminas/análise , Rodaminas/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
We report the synthesis, characterization, electrochemical sensing and catalytic capability of the bimetallic heterojunction Al2O3/ZnO micro flowers (AZ MFs). In order to prepare this bifunctional material, the facile hydrothermal process was adopted. The material was thoroughly characterized for the crystal structure and morphology with Powder XRD, XPS and FE-SEM. The investigation of electrochemical sensing was done using hydroquinone (HQ) and the chemical catalysis was using rhodamine B (RhB) with our bimetallic Al2O3/ZnO micro flowers as these are harmful industrial pollutants. The process parameters like the influence of scan rate and pH was efficiently optimized for the electrochemical detection of HQ and kinetics for the time dependent catalytic degradation of RhB dye. The linear relationship between the peak current and the concentration of HQ was found to be in the range of 0.125-20.25⯵M with an impressive detection limit of 11.2â¯nM. In the chemical catalytic degradation of the RhB dye, our bimetallic material thrived well during the reaction and degraded the material in 10â¯min. The performance of bimetallic Al2O3/ZnO micro flowers towards HQ detection and RhB degradation shows good stability, reproducibility and it can be efficiently utilized to treat the environmental pollutants.
Assuntos
Óxido de Alumínio/química , Técnicas Eletroquímicas/instrumentação , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Óxido de Zinco/química , Catálise , Eletrodos , Hidroquinonas/análise , Limite de Detecção , Modelos Teóricos , Reprodutibilidade dos Testes , Rodaminas/análise , Propriedades de Superfície , Purificação da Água/instrumentaçãoRESUMO
A high temperature desorption (HTD) direct analysis in real time-high-resolution mass spectrometric (DART-HRMS) method was developed for the rapid analysis of four banned cationic dyes. Rhodamine B is used to dye foods, while malachite green, crystal violet, and methylene blue are added to fishponds as antimicrobials. A simple induced phase separation extraction was used to pretreat samples. The DART-HRMS method employed two temperature steps, i.e., 200 °C for drying, purification, and enrichment of sample solution and 500 °C for thermal desorption and ionization of analytes. The calibration curves of dyes in the range of 50-2000 ng/mL were linear using deuterated malachite green as an internal standard. The LODs vary for all analytes between 0.1 and 30 ppb depending on the matrix and experimental conditions. Through analyses of real samples, two chili powders and one chili oil were found to be contaminated by rhodamine B. The concentrations were comparable with those found by an HPLC-MS/MS method.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Corantes/análise , Condimentos/análise , Contaminação de Alimentos/análise , Lagoas/análise , Rodaminas/análise , Espectrometria de Massas em Tandem/métodos , Limite de Detecção , Óleos de Plantas/análise , Especiarias/análise , Poluentes Químicos da Água/análiseRESUMO
The exploration of the relationship between zeolite composition and adsorption performance favored to facilitate its better application in removal of the hazardous substances from water. The adsorption capacity of rhodamine B (RB) onto Beta zeolite from aqueous solution was reported. The relationship between SiO2/Al2O3 ratio and adsorption capacity of Beta zeolite for RB was explored. The structure and physical properties of Beta zeolites with various SiO2/Al2O3 ratios were determined by XRD, FTIR, TEM, BET, UV-vis and so on characterizations. The adsorption behavior of rhodamine B onto Beta zeolite matched to Langmuir adsorption isotherm and more suitable description for the adsorption kinetics was a pseudo-second-order reaction model. The maximum adsorption capacity of the as-prepared Beta zeolite with SiO2/Al2O3 = 18.4 was up to 27.97mg/g.
Assuntos
Rodaminas/análise , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Zeolitas/química , Adsorção , Óxido de Alumínio/química , Cinética , Modelos Teóricos , Dióxido de Silício/química , Soluções , Propriedades de SuperfícieRESUMO
Rhodamine B was forbidden in food by law because of its carcinogenic properties to humans. However, due to its low cost, it was often used to dope chili oil by some counterfeiters to improve its natural color. However, it was difficult to quantify rhodamine B in chili oil due to its complex substrates and high viscosity. In this study, deep eutectic solvents, comprised of choline chloride and ethylene glycol, were first used as an extraction medium to separate rhodamine B from chili oil.
Assuntos
Formiatos/química , Óleos de Plantas/química , Rodaminas/análise , Água/química , Cromatografia Líquida de Alta Pressão , Estrutura Molecular , Solventes/química , Espectrometria de FluorescênciaRESUMO
A tracer test was conducted to aid in the investigation of water movement and solute transport at a crude-oil spill site near Bemidji, Minnesota. Time of travel was measured using breakthrough curves for rhodamine WT and bromide tracers moving from the soil surface through oil-contaminated and oil-free unsaturated zones to the saturated zone. Results indicate that the rates of tracer movement were similar in the oil-free unsaturated and saturated zones compared to the oily zones. These results are somewhat surprising given the oil contamination in the unsaturated and saturated zones. Rhodamine tracer breakthrough in the unsaturated and saturated zones in general was delayed in comparison to bromide tracer breakthrough. Peak tracer concentrations for the lysimeters and wells in the oily zone were much greater than at the corresponding depths in the oil-free zone. Water and tracer movement in the oily zone was complicated by soil hydrophobicity and decreased oil saturations toward the periphery of the oil. Preferential flow resulted in reduced tracer interaction with the soil, adsorption, and dispersion and faster tracer movement in the oily zone than expected. Tracers were freely transported through the oily zone to the water table. Recharge calculations support the idea that the oil does not substantially affect recharge in the oily zone. This is an important result indicating that previous model-based assumptions of decreased recharge beneath the oil were incorrect. Results have important implications for modeling the fate and transport of dissolved contaminants at hydrocarbon spill sites.
Assuntos
Água Subterrânea/análise , Hidrologia/métodos , Poluição por Petróleo , Solo , Poluentes Químicos da Água/análise , Brometos/análise , Água Subterrânea/química , Minnesota , Modelos Teóricos , Petróleo/análise , Rodaminas/análise , Movimentos da ÁguaRESUMO
In excitation-emission fluorescence spectroscopy, the simultaneous quantitative prediction and qualitative resolution of mixtures of fluorophores using chemometrics is a major challenge because of the scattering and reabsorption effects (turbidity) presented mainly in biomaterials. The measured fluorescence spectra are distorted by multiple scattering and reabsorption events in the surrounding medium, thereby diminishing the performance of the commonly used three-way resolution methods such as parallel factor (PARAFAC) analysis or multivariate curve resolution-alternating least squares (MCR-ALS). In this work we show that spectral loadings and concentration profiles from model mixtures provided using PARAFAC and MCR-ALS are severely distorted by reabsorption and scattering phenomena, although both models fit rather well the experimental data in terms of percentage of the explained variance. The method to correct the fluorescence excitation-emission matrix (EEM) consisted in measuring the optical properties (absorption parameter µa , scattering parameter µs, and anisotropy factor g) of samples and calculating the corresponding transfer function by means of the Monte Carlo simulation method. By applying this transfer function to the measured EEM, it was possible to compensate for reabsorption and scattering effects and to restore the ideal EEM, i.e., the EEM that is due only to fluorophores, without distortions from the absorbers and scatterers that are present. The PARAFAC and MCR-ALS decomposition of the resulting ideal EEMs provided spectral loadings and concentration profiles that matched the true profiles.
Assuntos
Corantes Fluorescentes/análise , Espectrometria de Fluorescência/métodos , Absorção de Radiação , Algoritmos , Anisotropia , Simulação por Computador , Emulsões/química , Amarelo de Eosina-(YS)/análise , Fluoresceína/análise , Análise dos Mínimos Quadrados , Método de Monte Carlo , Fosfolipídeos/química , Quinolinas/química , Rodaminas/análise , Espalhamento de Radiação , Soluções , Óleo de Soja/química , Espectrometria de Fluorescência/instrumentaçãoRESUMO
Cardiac hypertrophy is the main response of the heart to various extrinsic and intrinsic stimuli, and it is characterized by specific molecular and phenotypic changes. Recent in vitro and in vivo studies indicate the involvement of reactive oxygen species in the hypertrophic response. In this study, silibinin, a plant flavonolignan extracted from milk thistle with potent antioxidant activity, was evaluated for its effects in (a) preventing hydrogen peroxide (H2O2)-induced cellular damage and (b) blocking the phenylephrine-induced hypertrophic response. Using the in vitro model of embryonic rat heart-derived H9c2 cells, we showed that silibinin has a rather safe profile as concentrations up to 200µM did not affect cell viability. Pretreatment of H9c2 cells with silibinin resulted in better protection of H9c2 cells under conditions of H2O2-induced cellular stress compared to untreated cells as indicated by cell viability and DNA fragmentation assays. Furthermore, silibinin attenuated the phenylephrine-induced hypertrophic response as evidenced by the measurement of cell surface, up-regulation of atrial natriuretic peptide and increase of cellular protein levels. Moreover, silibinin repressed the phenylephrine-induced phosphorylation of ERK1/2 kinases, while it appeared to inhibit the weakly activated by phenylephrine phosphorylation of Akt. Based on our results, silibinin may attenuate the phenylephrine-induced hypertrophic response of H9c2 cells via antioxidant mechanisms involving mainly the inhibition of the intracellular signaling pathways mediated by ERK1/2 MAPKs and Akt.
Assuntos
Cardiomegalia/tratamento farmacológico , Coração/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fenilefrina/efeitos adversos , Extratos Vegetais/farmacologia , Silimarina/farmacologia , Animais , Antioxidantes , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Sobrevivência Celular , Fragmentação do DNA , Coração/fisiologia , Peróxido de Hidrogênio/efeitos adversos , Peróxido de Hidrogênio/metabolismo , Silybum marianum/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodaminas/análise , Rodaminas/metabolismo , Transdução de Sinais , Silibina , Regulação para CimaRESUMO
Surface enhanced Raman scattering (SERS) is sensitive enough for single-molecule biochemical detection, but it is extremely difficult to obtain a large number of SERS hotspots for sensitive and reproducible detection. It is even more challenging to assemble the hotspots at designated positions for location predictable sensing. Here, we report an original strategy for the synthesis, manipulation, and assembling of plasmonic nanocapsule SERS sensors for high-sensitivity biochemical detection at predictable locations.
Assuntos
Nanocápsulas/química , Óxido de Alumínio/química , Eletrodos , Galvanoplastia , Magnetismo , Nanopartículas Metálicas/química , Porosidade , Rodaminas/análise , Prata/química , Análise Espectral RamanRESUMO
Argonaute proteins are the core components of the microRNP/RISC. The biogenesis and function of microRNAs and endo- and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA binding and nuclease activities. Currently, there are no in vitro assays suitable for large-scale screening of microRNP/RISC loading modulators. We describe a novel in vitro assay that is based on fluorescence polarization of TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we identified potent small-molecule inhibitors of RISC loading, including aurintricarboxylic acid (IC(50) = 0.47 µM), suramin (IC(50) = 0.69 µM), and oxidopamine HCL (IC(50) = 1.61 µM). Small molecules identified by this biochemical screening assay also inhibited siRNA loading to endogenous Ago2 in cultured cells.
Assuntos
Proteínas Argonautas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Complexo de Inativação Induzido por RNA/antagonistas & inibidores , RNA/análise , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular , DNA/metabolismo , Corantes Fluorescentes/análise , Humanos , RNA/metabolismo , RNA Interferente Pequeno/antagonistas & inibidores , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Rodaminas/análiseRESUMO
For phosphorus (P) transport from upland areas to surface water systems, the primary transport mechanism is typically considered to be surface runoff with subsurface transport assumed negligible. However, certain local conditions can lead to an environment where subsurface transport may be significant. The objective of this research was to determine the potential of subsurface transport of P along streams characterized by cherty or gravel subsoils, especially the impact of preferential flow paths on P transport. At a field site along the Barren Fork Creek in northeastern Oklahoma, a trench was installed with the bottom at the topsoil/alluvial gravel interface. Fifteen piezometers were installed surrounding the trench to monitor flow and transport. In three experiments, water was pumped into the trench from the Barren Fork Creek to maintain a constant head. At the same time, a conservative tracer (Rhodamine WT) and/or potassium phosphate solution were injected into the trench at concentrations at 3 and 100 mg/L for Rhodamine WT and at 100 mg/L for P. Laboratory flow-cell experiments were also conducted on soil material <2 mm in size to determine the effect that flow velocity had on P sorption. Rhodamine WT and P were detected in some piezometers at equivalent concentrations as measured in the trench, suggesting the presence of preferential flow pathways and heterogeneous interaction between streams and subsurface transport pathways, even in nonstructured, coarse gravel soils. Phosphorus transport was retarded in nonpreferential flow paths. Breakthrough times were approximately equivalent for Rhodamine WT and P suggesting no colloidal-facilitated P transport. Results from laboratory flow-cell experiments suggested that higher velocity resulted in less P sorption for the alluvial subsoil. Therefore, differences in flow rates between preferential and nonpreferential flow pathways in the field led to variable sorption. The potential for nutrient subsurface transport shown by this alluvial system has implications regarding management of similar riparian floodplain systems.
Assuntos
Fósforo/análise , Solo/análise , Poluentes Químicos da Água/análise , Poluição Química da Água/análise , Ecossistema , Oklahoma , Rodaminas/análiseRESUMO
Emission and excitation spectra of 3- and 4-pyridinecarboxaldehyde vapors have been measured at different pressures down to 10(-2)Torr. The phosphorescence quantum yield measured at low pressure as a function of excitation energy is nearly constant in the range of excitation energy corresponding to the S1(n, pi*) state, but it decreases abruptly at the S2(pi, pi*) threshold. The onset of the abrupt decrease of the yield corresponds to the location of the S2 absorption origin of each molecule, indicating that the nonradiative pathway depends on the type of the excited singlet state to which the molecule is initially excited. The relaxation processes are discussed based on the pressure and excitation-energy dependence of the phosphorescence quantum yield.
Assuntos
Fósforo/análise , Fotoquímica/métodos , Piridinas/química , Cinética , Pressão , Teoria Quântica , Rodaminas/análise , Espectrofotometria/métodosRESUMO
A portable luminescence spectrofluorometer weighing only 1.5 kg that uses multiple light emitting diodes (LEDs) as excitation sources was developed and evaluated. Excitation using a sequence of seven individual broad-band LED emission sources enabled the generation of excitation-emission spectra using a light weight (<1.5 kg) spectrometer. Limits of detection for rhodamine 6G, rhodamine B, and fluorescein were 2.9, 3.2, and 11.0 nM, respectively. Generation of excitation-emission matrices (EEMs) enabled the analysis of samples containing mixtures of rhodamine B and fluorescein. Buffered saline plant and animal feed extracts were also analyzed using this instrument. These samples included the woody plants Pistacia lentiscus (Evergreen pistache or Mastic) and Philyria latifolia, and the herbaceous species Medicago sativa (alfalfa), Trifolium spp. (clover), and a feed concentrate. Application of multi-way principal component analysis (MPCA) to the resulting three-dimensional data sets enabled discernment among these various diet constituents.
Assuntos
Semicondutores , Ração Animal/análise , Animais , Desenho de Equipamento , Luz , Iluminação/instrumentação , Iluminação/métodos , Extratos Vegetais/química , Análise de Componente Principal , Rodaminas/análise , Riboflavina/análise , Espectrometria de Fluorescência/instrumentaçãoRESUMO
Since the major approach in searching for potential anticancer agents over the last 50 years has been based on selective cytotoxic effects on mammalian cancer cell lines, cell-based methods for cytotoxicity are described and compared. The sulphorhodamine B (SRB) assay is described in detail as the preferred method and also a novel approach has been developed which is based on the hypothesis that, in some circumstances, the naturally occurring compounds act as prodrugs rather than active compounds in their own right. Consequently, extracts or compounds are pre-incubated with systems modelling metabolic processes in the body before being tested. The methods have been validated using known compounds and Iris tectorum extracts have been shown to be more cytotoxic after treatment with beta-glucosidase. In addition bioassays based on mammalian cells involving antioxidant and upregulation of some cellular self-defence mechanisms are discussed which are related to prevention as well as treatment of cancer. Extracts of Alpinia officinarum induced glutathione-S-transferase (GST) activity in cultured hepatocytes and this was traced to the phenylpropanoids present, especially 1'-acetoxychavicol acetate.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Plantas Medicinais/química , Plantas Medicinais/classificação , Pró-Fármacos/química , Rodaminas/química , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Glutationa Transferase/efeitos dos fármacos , Glutationa Transferase/metabolismo , Humanos , Estrutura Molecular , Pró-Fármacos/análise , Rodaminas/análise , Rodaminas/metabolismo , Regulação para CimaRESUMO
A combined surface sampling probe/electrospray emitter coupled with an ion trap mass spectrometer was used for the direct read out of unmodified reversed-phase C18 thin-layer chromatography (TLC) plates. The operation of the surface sampling electrospray ionization interface in positive and negative ionization modes was demonstrated through the direct analysis of TLC plates on which a commercial test mix comprised of four dye compounds viz., rhodamine B, fluorescein, naphthol blue black, and fast green FCF, and an extract of the caffeine-containing plant Ilex vomitoria, were spotted and developed. Acquisition of full-scan mass spectra and automated collection of MS/MS product ion spectra while scanning a development lane along the surface of a TLC plate demonstrated the advantages of using an ion trap in this combination. Details of the sampling system, benefits of analyzing a developed lane in both positive ion and negative ion modes, levels of detection while surface scanning, surface scan speed effects, and the utility of three-dimensional data display, are also discussed.
Assuntos
Cromatografia em Camada Fina/instrumentação , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Negro de Amido/análise , Cromatografia em Camada Fina/métodos , Corantes/análise , Fluoresceína/análise , Ilex vomitoria/química , Corantes Verde de Lissamina/análise , Extratos Vegetais/análise , Extratos Vegetais/química , Rodaminas/análise , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
We investigated cellular trafficking of dermal macrophages that express a macrophage calcium-type lectin (MMGL) during the sensitization of delayed-type hypersensitivity. In skin, dermal macrophages, but not epidermal Langerhans cells, have been shown to express MMGL. Epicutaneous sensitization by FITC produced a transient increase in MMGL-positive cells in regional lymph nodes. To directly investigate whether the increase was due to cell migration from dermis, MMGL-positive cells purified from skin were intradermally injected into syngeneic mice after labeling with a fluorescent cell tracer, followed by epicutaneous sensitization over the site of injection. MMGL-positive cells containing the tracer were found in the regional lymph nodes after sensitization. The majority of the MMGL-positive cell migrants were negative for FITC fluorescence despite the presence of FITC-labeled cells that included Langerhans cell migrants. Because the extent of MMGL-positive cell migration was greatly influenced by the selection of vehicles to dissolve FITC, the efficiency of sensitization was compared using the ear swelling test. Migration of both Langerhans cells (FITC-labeled cells) and MMGL-positive cells contributed positively to the efficiency of sensitization. Interestingly, MMGL-positive cell migration was induced by vehicle alone, even in the absence of FITC. These results suggest that migration of dermal MMGL-positive cells accounts for the adjuvant effects of vehicles at least in part.
Assuntos
Dermatite Alérgica de Contato/patologia , Lectinas Tipo C , Macrófagos/fisiologia , Proteínas de Membrana , Pele/patologia , Adjuvantes Imunológicos , Administração Cutânea , Animais , Assialoglicoproteínas , Biomarcadores , Proteínas de Transporte/análise , Movimento Celular , Feminino , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Células de Langerhans/patologia , Lectinas/análise , Linfonodos/patologia , Macrófagos/classificação , Camundongos , Camundongos Endogâmicos BALB C , Rodaminas/análise , Organismos Livres de Patógenos EspecíficosRESUMO
BACKGROUND: It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles. RESULTS: In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1-adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells. Thus, specific changes in intracellular Ca2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca2+ indicator. CONCLUSION: GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters.
Assuntos
Células Eucarióticas/ultraestrutura , Corantes Fluorescentes , Proteínas Luminescentes , Mitocôndrias/metabolismo , Organelas/ultraestrutura , Proteínas Recombinantes de Fusão , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Biomarcadores , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Citosol/química , DNA Complementar/genética , Células Eucarióticas/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/efeitos da radiação , Expressão Gênica , Proteínas de Fluorescência Verde , Células HeLa/química , Células HeLa/ultraestrutura , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Histamina/farmacologia , Humanos , Luz , Medições Luminescentes , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Proteínas Luminescentes/efeitos da radiação , Mitocôndrias/efeitos dos fármacos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Rodamina 123 , Rodaminas/análise , Cifozoários/química , Cifozoários/genética , Transfecção , Raios UltravioletaRESUMO
Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that localizes preferentially within mitochondria of living carcinoma cells. MGH-U1 human bladder carcinoma cells incubated in vitro with 10 microM R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser underwent selective, phototoxic injury to mitochondria. Ultrastructurally, treatment with R123 plus irradiation with 10 J/cm2 caused selective, progressive mitochondrial alterations consisting of disruption of cristae, vacuolization, swelling, increasing numbers of ring-shaped and angulated mitochondria at 4 to 8 h after irradiation, and obliteration of many mitochondria at 24 to 48 h. Confocal laser scanning microscopy after treatment with R123 plus irradiation with 10 to 30 J/cm2 demonstrated altered uptake and localization of subsequently administered R123, accompanied by striking mitochondrial fragmentation. Irradiation caused a dose-dependent depletion of extractable R123, due to a photosensitized efflux that began immediately and progressed by 4 h after irradiation with 10 to 30 J/cm2; further uptake after reincubation in the presence of R123 was also quantitatively impaired in cells previously irradiated with 30 J/cm2.