RESUMO
To mimic more realistic lung tissue conditions, co-cultures of epithelial and immune cells are one comparatively easy-to-use option. To reveal the impact of immune cells on the mode of action (MoA) of CuO nanoparticles (NP) on epithelial cells, A549 cells as a model for epithelial cells have been cultured with or without differentiated THP-1 cells, as a model for macrophages. After 24 h of submerged incubation, cytotoxicity and transcriptional toxicity profiles were obtained and compared between the cell culture systems. Dose-dependent cytotoxicity was apparent starting from 8.0 µg/cm2 CuO NP. With regard to gene expression profiles, no differences between the cell models were observed concerning metal homeostasis, oxidative stress, and DNA damage, confirming the known MoA of CuO NP, i.e., endocytotic particle uptake, intracellular particle dissolution within lysosomes with subsequent metal ion deliberation, increased oxidative stress, and genotoxicity. However, applying a co-culture of epithelial and macrophage-like cells, CuO NP additionally provoked a pro-inflammatory response involving NLRP3 inflammasome and pro-inflammatory transcription factor activation. This study demonstrates that the application of this easy-to-use advanced in vitro model is able to extend the detection of cellular effects provoked by nanomaterials by an immunological response and emphasizes the use of such models to address a more comprehensive MoA.
Assuntos
Epitélio/efeitos dos fármacos , Nanopartículas Metálicas/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Estresse Oxidativo/genética , Transcrição Gênica/efeitos dos fármacos , Células A549 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Cobre/química , Cobre/farmacologia , Dano ao DNA/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: We previously demonstrated that conditional overexpression of neuronal nitric oxide synthase (nNOS) inhibited L-type Ca2+ channels and decreased myocardial contractility. However, nNOS has multiple targets within the cardiac myocyte. We now hypothesize that nNOS overexpression is cardioprotective after ischemia/reperfusion because of inhibition of mitochondrial function and a reduction in reactive oxygen species generation. METHODS AND RESULTS: Ischemia/reperfusion injury in wild-type mice resulted in nNOS accumulation in the mitochondria. Similarly, transgenic nNOS overexpression caused nNOS abundance in mitochondria. nNOS translocation into the mitochondria was dependent on heat shock protein 90. Ischemia/reperfusion experiments in isolated hearts showed a cardioprotective effect of nNOS overexpression. Infarct size in vivo was also significantly reduced. nNOS overexpression also caused a significant increase in mitochondrial nitrite levels accompanied by a decrease of cytochrome c oxidase activity. Accordingly, O(2) consumption in isolated heart muscle strips was decreased in nNOS-overexpressing nNOS(+)/αMHC-tTA(+) mice already under resting conditions. Additionally, we found that the reactive oxygen species concentration was significantly decreased in hearts of nNOS-overexpressing nNOS(+)/αMHC-tTA(+) mice compared with noninduced nNOS(+)/αMHC-tTA(+) animals. CONCLUSION: We demonstrated that conditional transgenic overexpression of nNOS resulted in myocardial protection after ischemia/reperfusion injury. Besides a reduction in reactive oxygen species generation, this might be caused by nitrite-mediated inhibition of mitochondrial function, which reduced myocardial oxygen consumption already under baseline conditions.
Assuntos
Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/metabolismo , Modelos Animais , Infarto do Miocárdio/patologia , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
We present a new probe for the determination of hydrogen peroxide (HP). It is based on the yellow fluorophore 4-amino-1,8-napththalimide, coupled to p-anisidine (as a redox-active group) to form a probe that is based on photoinduced electron transfer (PET). The preparation of the probe (which we refer to as "HP Green") was accomplished in four steps with good yield. Its fluorescence is independent of pH in the physiological range and quenched by a PET process that occurs between the p-anisidine redox moiety and the naphthalimide luminophore. If the p-anisidine group is oxidized by HP, PET is suppressed and fluorescence intensity is strongly increased. Addition of horseradish peroxidase (HRP) enhances the oxidation of HP Green and further improves the detection limit of HP. The use of HRP and HP Green enables the determination of HP concentration in a range of 0.1 to 5 µM, with a limit of detection (LOD) as low as 64 nM (16 pmol per well in microtiter plates). HP Green and HRP also enable sensitive enzymatic assays of oxidase substrates in a kinetic format, as shown for L-lactate and D-glucose. L-Lactate concentration can be rapidly determined between 0.5 and 10 µM after 6 minutes of incubation at 30 °C, with an LOD of 164 nm (41 pmol per well). This LOD is more than sixfold lower than that of the best commercial assays for lactate. The detection range for D-glucose is 2 to 30 µm, and the LOD is 644 nM (161 pmol per well). These are among the lowest concentrations detectable for oxidase-based assays. The hexanoic acid moiety in HP Green may be further used to immobilize the probe in order to obtain sensor layers for continuous assays.
Assuntos
Corantes Fluorescentes , Glucose/análise , Peróxido de Hidrogênio/química , Ácido Láctico/análise , Tomografia por Emissão de Pósitrons , Cinética , Limite de Detecção , Espectrometria de FluorescênciaRESUMO
Novel amino-reactive phenoxazines were obtained by reasonably simple synthetic protocols and characterized in terms of their use as fluorescent labels for amines, amino acids and proteins in general. Purple labels (alternatives to Texas Red) and blue labels (alternatives to Cy-1) were obtained by this strategy. The absorption/emission maxima, in aqueous solution, are at around 589/630 nm and 648/670 nm, respectively, thus indicating larger Stokes' shifts than those of common cyanine-type of labels. The new labels are compatible with commercial diode laser light sources.
Assuntos
Aminas/química , Corantes Fluorescentes/química , Oxazinas/química , Proteínas/química , Xantenos/química , Aminoácidos/química , Corantes Fluorescentes/síntese química , Estrutura Molecular , Oxazinas/síntese química , EstereoisomerismoRESUMO
While the toxicity of metal-based nanoparticles (NP) has been investigated in an increasing number of studies, little is known about metal-based fibrous materials, so-called nanowires (NWs). Within the present study, the physico-chemical properties of particulate and fibrous nanomaterials based on Cu, CuO, Ni, and Ag as well as TiO2 and CeO2 NP were characterized and compared with respect to abiotic metal ion release in different physiologically relevant media as well as acellular reactivity. While none of the materials was soluble at neutral pH in artificial alveolar fluid (AAF), Cu, CuO, and Ni-based materials displayed distinct dissolution under the acidic conditions found in artificial lysosomal fluids (ALF and PSF). Subsequently, four different cell lines were applied to compare cytotoxicity as well as intracellular metal ion release in the cytoplasm and nucleus. Both cytotoxicity and bioavailability reflected the acellular dissolution rates in physiological lysosomal media (pH 4.5); only Ag-based materials showed no or very low acellular solubility, but pronounced intracellular bioavailability and cytotoxicity, leading to particularly high concentrations in the nucleus. In conclusion, in spite of some quantitative differences, the intracellular bioavailability as well as toxicity is mostly driven by the respective metal and is less modulated by the shape of the respective NP or NW.
RESUMO
In this study, we present a novel amino-reactive fluorescence marker (referred to as UR-431), which is well suited for electrophoretic techniques. A main feature of this marker is its weakly basic behavior when conjugated to analytes. Labeled primary amines exhibit a positive net charge and accordingly a cathodic mobility below a pH of 2.4. The label features a pH-independent fluorescence emission and is thus very interesting for electrophoretic applications such as IEF. The absorption maximum of this yellow daylight chromophore is at 431 nm, whereas fluorescence emission peaks at 537 nm (quantum yield approximately 0.1). The label was successfully conjugated to amines, peptides and proteins and separated via CE and MCE. The on-chip detection limit of labeled lysine using a mercury-lamp-based fluorescence microscope was determined as 12 nM. An important feature of the new label is that it effects only a subtle change of the pI of proteins compared with common anionic labels, e.g. FITC. pI values of proteins were investigated by comparing native proteins and labeled proteins in CIEF. UR-431 was also applied to sensitive detection of amines and peptides in MCE.
Assuntos
Peptídeos/química , Proteínas/química , Aminoácidos/análise , Aminoácidos/isolamento & purificação , Eletroforese/métodos , Eletroforese Capilar/métodos , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Indicadores e Reagentes , Focalização Isoelétrica/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
A method is described for modifying the surface of upconverting microparticles (UCmuPs) and nanoparticles (UCNPs) such that they become amenable to click chemistry. Respective reagents are presented and used in both kinds of particles, either directly or in combination with tetraethoxysilane. The particles also were labeled by using the click reaction, a) with fluorophores to yield materials that have emission colors that depend on the wavelength of excitation; b) with maleinimido groups (so to obtain labels for thiols), and c) with biotin (to make them useful for affinity studies based on the biotin-streptavidin system). We believe that both the UCmuPs and UCNPs have the potential of being used in numerous areas including upconversion imaging, biolabeling, and derivatization, but also in encoding and security.
RESUMO
The synthesis of a set of new clickable fluorophores that virtually cover the whole visible spectrum reaching the near infra-red regime is presented herein. Besides dyes that are capable of participating in classical copper catalyzed 1,3-dipolar cycloaddition reactions with the counterparting function we have also prepared dyes containing a cyclooctyne moiety, an alkyne derivative that enables copper free clicking to azides. The suitability of these dyes for fluorescent labeling of biomolecules is presented by examples on model frameworks representing major biopolymer building blocks. The versatility of these dyes is presented in cell labeling experiments as well as by labeling the azide modified surface glycans of CHO-cells either by copper catalyzed or copper-free click reaction. These dyes are expected to have a large variety of applications in (bio)orthogonal labeling schemes both in vivo and in vitro.
Assuntos
Cobre/química , Corantes Fluorescentes/síntese química , Animais , Células CHO , Cricetinae , Cricetulus , Ciclo-Octanos/química , Cicloparafinas/química , Técnicas de Sonda Molecular , Polissacarídeos/químicaRESUMO
We report on the preparation of fluorescent silica nanoparticles (NPs). They have been prepared by (a) modification of the NPs by amino groups and subsequent introduction of amino-reactive fluorophores of various color and (b) by modification of the NPs by either azido groups or alkyne groups and subsequent conjugation to fluorophores by so-called click chemistry, which is a novel approach toward modifying silica NPs. The new NPs were characterized in terms of size and spectral properties.