RESUMEN
Triphenylamine-sensitized 8-dimethylaminoquinoline (TAQ) probes showed fair two-photon absorption and fragmentation cross sections in releasing kainate and GABA ligands. The water-soluble PEG and TEG-analogs allowed cell internalization and efficient light-gated liberation of the rhodamine reporter under UV and two-photon (NIR) irradiation conditions.
RESUMEN
Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.
Asunto(s)
Aminas/toxicidad , Endocitosis/efectos de los fármacos , Endocitosis/efectos de la radiación , Espacio Intracelular/metabolismo , Luz , Aminas/química , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Células HeLa , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de la radiación , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Nitric-oxide synthases (NOS) catalyze the formation of NO using NADPH as electron donor. We have recently designed and synthesized a new series of two-photon absorbing and photoactivatable NADPH analogues (NT). These compounds bear one or two carboxymethyl group(s) on the 2'- or/and 3'-position(s) of the ribose in the adenosine moiety, instead of a 2'-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Here, we addressed the ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells. METHODS: The cellular fate of NTs and their photoinduced effects were studied using multiphoton fluorescence imaging, cell viability assays and a BODIPY-derived NO probe for NO measurements. The eNOS dependence of photoinduced NO production was addressed using two NOS inhibitors (NS1 and L-NAME) targeting the reductase and the oxygenase domains, respectively. RESULTS: We found that, two compounds, those bearing a single carboxymethyl group on the 3'-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation was observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects. CONCLUSIONS: We show for the first time that NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells. GENERAL SIGNIFICANCE: Efficient photoactivatable NADPH analogues targeting NOS could have important implications for generating apoptosis in tumor cells or modulating NO-dependent physiological processes.
Asunto(s)
Aparato de Golgi/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Luz , NADP , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Humanos , NADP/análogos & derivados , NADP/farmacologíaRESUMEN
Photodynamic therapy (PDT) is a promising therapeutic method for several diseases, in particular for cancer. This approach uses a photosensitizer, oxygen, and an external light source to produce reactive oxygen species (ROS) at lethal doses to induce cell death. One drawback of current PDT is the use of visible light which has poor penetration in tissues. Such a limitation could be overcome by the use of novel organic compounds compatible with photoactivation under near-infrared light excitation. Triphenylamines (TPAs) are highly fluorescent compounds that are efficient to induce cell death upon visible light excitation (458 nm), but outside the biological spectral window. Interestingly, we recently showed that TPAs target cytoplasmic organelles of living cells, mainly mitochondria, and induce a high ROS production upon 2-photon excitation (in the 760-860 nm range), leading to a fast apoptosis process. However, we observed significant differences among the tested TPA compounds in terms of cell distribution and time courses of cell death-related events (apoptosis vs necrosis). In summary, TPAs represent serious candidates as photosensitizers that are compatible with 2-photon excitation to simultaneously trigger and imaging cell death although the relationship between their subcellular localization and the cell death mechanism involved is still a matter of debate.
Asunto(s)
Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/química , Apoptosis/fisiología , Muerte Celular/fisiología , Humanos , Imagen Óptica/métodos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Photodynamic therapy (PDT) leads to cell death by using a combination of a photosensitizer and an external light source for the production of lethal doses of reactive oxygen species (ROS). Since a major limitation of PDT is the poor penetration of UV-visible light in tissues, there is a strong need for organic compounds whose activation is compatible with near-infrared excitation. Triphenylamines (TPAs) are fluorescent compounds, recently shown to efficiently trigger cell death upon visible light irradiation (458 nm), however outside the so-called optical/therapeutic window. Here, we report that TPAs target cytosolic organelles of living cells, mainly mitochondria, triggering a fast apoptosis upon two-photon excitation, thanks to their large two-photon absorption cross-sections in the 760-860 nm range. Direct ROS imaging in the cell context upon multiphoton excitation of TPA and three-color flow cytometric analysis showing phosphatidylserine externalization indicate that TPA photoactivation is primarily related to the mitochondrial apoptotic pathway via ROS production, although significant differences in the time courses of cell death-related events were observed, depending on the compound. TPAs represent a new class of water-soluble organic photosensitizers compatible with direct two-photon excitation, enabling simultaneous multiphoton fluorescence imaging of cell death since a concomitant subcellular TPA re-distribution occurs in apoptotic cells.
Asunto(s)
Compuestos de Anilina/metabolismo , Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Imagen Óptica/métodos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Muerte Celular , Citometría de Flujo , Células HeLa , Humanos , Luz , Especies Reactivas de Oxígeno/análisisRESUMEN
Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX(4) and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX(4) -associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Luz , Melanoma/metabolismo , NADP/farmacología , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Apoptosis , Western Blotting , Ciclo Celular , Proliferación Celular , Espectroscopía de Resonancia por Spin del Electrón , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , NADP/análogos & derivados , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Neovascularización Patológica , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We implement three photon fluorescence polarization resolved microscopy to optically investigate molecular and protein crystals. The availability of UV transitions using IR pulses allows analyses without fluorescence staining. Polarization resolved studies indicate that high-order symmetry structures can be revealed and that strong fluorescent energy transfer occurs between molecules. We show how this permits identification of a monocrystalline nature for a sample at the subwavelength resolution scale.
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Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Modelos Químicos , Proteínas/química , Animales , Cristalización , Proteínas del Huevo/química , Transferencia Resonante de Energía de Fluorescencia , Microscopía de Polarización/métodos , Muramidasa/químicaRESUMEN
We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.
Asunto(s)
Colorantes Fluorescentes , Células Endoteliales de la Vena Umbilical Humana/enzimología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , NADP , Óxido Nítrico Sintasa de Tipo III , Catálisis , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Células Endoteliales de la Vena Umbilical Humana/química , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Simulación de Dinámica Molecular , NADP/análogos & derivados , NADP/síntesis química , NADP/química , NADP/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico/química , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismoRESUMEN
Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions.
Asunto(s)
Decápodos , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Secuencia de Aminoácidos , Alimentación Animal , Animales , Secuencia de Bases , Polarización de Fluorescencia , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Amorphous red-emitting materials involving solvatochromic small molecules have been processed by the reprecipitation method as non-doped nanospheres characterized by a remarkably low polydispersity. Their mean diameter could simply be tuned by the concentration of the organic solution giving rise to time-stable dispersion of 85-200 nm-sized nanoparticles. Time-resolved measurements performed on solid nanoparticles showed significant size-dependence effects of the emission lifetime and maxima evidencing populations with distinct molecular conformations. Nanoparticle internalization has proved successful in NIH-3T3 murine fibroblasts with normal toxicity effects after 48 h. Fluorescence confocal microscopy under one- and two-photon excitations revealed dual emission enabling localization of the organic material within the plasma membrane and the cytoplasm. Model experiments resorting to suspended artificial lipid bilayers allowed us to conclude on the dissolution of nanoparticles by the phospholipid membrane during the internalization process. They let us to assume that uptake of hydrophobic nanoparticles by living cells implies an endocytosis mechanism operating through the formation of plasmic vesicles.
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Colorantes Fluorescentes/química , Membrana Dobles de Lípidos/química , Nanopartículas/química , Compuestos Orgánicos/química , Animales , Cápsulas/química , Línea Celular , Supervivencia Celular , Liposomas/química , Ratones , Microscopía Electrónica de Transmisión , Modelos Biológicos , Compuestos Orgánicos/síntesis química , Tamaño de la Partícula , Espectrometría de FluorescenciaRESUMEN
We report on the investigation of the structure of DNA liquid crystal (LC) phases by means of polarization sensitive two-photon microscopy (PSTPM). DNA was stained with fluorescent dyes, an intercalator propidium iodide, or a groove binder Hoechst 3342, and the angular dependence of the intensity of two-photon excited fluorescence emitted by the dye was collected. The local orientation of DNA molecules in cholesteric and columnar LC phases was established on the basis of the relative angle between the transition dipole of the dye and the long axis of DNA helix. Three-dimensional images of the cholesteric phase were obtained making use of the intrinsic 3D resolving ability of two-photon microscopy. We also discuss the influence of dyes on the parameters of DNA LC phases and comment on advantages and limitations of the PSTPM technique in comparison with other LC characterization techniques.
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ADN/química , Cristales Líquidos/química , Imagenología Tridimensional , Sustancias Intercalantes/química , Microscopía Fluorescente/métodos , Microscopía de Polarización/métodos , Fotones , Propidio/química , SolucionesRESUMEN
We have recently reported the analytical performance of an immunosensor comprising one mm-scale parallel plate laminar flow chamber and applied to capture MCF7 breast cancer cells (Ehrhart et al., Biosens. Bioelectr. 24, 467, 2008). Herein we present a new multiplex immunosensor embodying four parallel plate laminar flow chambers that fit onto a standard, functionalized, microscopy glass slide. The four surfaces are coated with long alkyl chain spacers of 21-aminohenicosyl trichlorosilane (AHTS) and then grafted with a monoclonal anti-human epithelial cell adhesion molecule (EpCAM) antibody specific of target cells to immobilize. We first demonstrate a significantly (P < 0.01) improved capacity of each of the four flow chambers of the multiplex immunosensor to capture MCF7 cells compared to the previous single chamber device. Second, in addition to an increase of cell immobilization, the multiplex device offers a versatile tool easily grafted with various purified antibodies onto the four surfaces. Third, we obtained high cell capture rate and efficiency of various numbers of MCF7 cells spiked in buffer containing an equal number of background leukocytes. And fourth, we demonstrate isolation efficiency of circulating tumor cells (CTCs) from peripheral blood drawn from a small cohort of patients with localized or metastatic breast cancer. This new multiplex immunosensor could be tested for its potential to capture different subpopulations of CTCs.
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Técnicas Biosensibles/instrumentación , Neoplasias de la Mama/sangre , Separación Celular/instrumentación , Inmunoensayo/instrumentación , Queratinas/metabolismo , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Anticuerpos Inmovilizados/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Vidrio/química , Humanos , Queratinas/sangreRESUMEN
UV-assisted photocleavage in the solid state of orange emitting nitro-substituted triarylamines leads to the appearance of blue emission following photodisruption of the ICT state.
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Aminas/química , Fotólisis , Espectrometría de Fluorescencia , Rayos UltravioletaRESUMEN
Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size <40 nm), resonant laser scattering and Raman scattering cross sections are too small to allow single nanoparticle observation. Nanodiamonds can, however, be rendered photoluminescent with a perfect photostability at room temperature. Such a remarkable property allows easier single-particle tracking over long time scales. In this work, we use photoluminescent nanodiamonds of size <50 nm for intracellular labeling and investigate the mechanism of their uptake by living cells. By blocking selectively different uptake processes, we show that nanodiamonds enter cells mainly by endocytosis, and converging data indicate that it is clathrin-mediated. We also examine nanodiamond intracellular localization in endocytic vesicles using immunofluorescence and transmission electron microscopy. We find a high degree of colocalization between vesicles and the biggest nanoparticles or aggregates, while the smallest particles appear free in the cytosol. Our results pave the way for the use of photoluminescent nanodiamonds in targeted intracellular labeling or biomolecule delivery.
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Cristalización/métodos , Diamante/farmacocinética , Mediciones Luminiscentes/métodos , Nanoestructuras/química , Nanoestructuras/ultraestructura , Nanotecnología/métodos , Diamante/química , Células HeLa , Humanos , Sustancias Macromoleculares/química , Ensayo de Materiales , Conformación Molecular , Tamaño de la Partícula , Coloración y Etiquetado/métodos , Propiedades de SuperficieRESUMEN
Kit is a cell surface type III tyrosine kinase (TK) receptor implicated in cell transformation through overexpression or oncogenic mutation. Two categories of Kit mutants displaying mutations either in the juxtamembrane intracellular domain (regulatory mutants) or in the catalytic domain (catalytic mutants) have been described. To explore the effect of Kit oncogenic mutations on its subcellular localization, we constructed enhanced green fluorescent protein (EGFP)-tagged human Kit chimeras harboring mutations either in the regulatory (V560G) or in the catalytic (D816V) domain. When expressed in Chinese hamster ovary cells, EGFP-tagged wild-type Kit was activated on stem cell factor stimulation, whereas both EGFP-tagged Kit mutants displayed a constitutive TK activity. Constitutively activated mutants exhibited a high-mannose-type N-glycosylation pattern and an intracellular localization, suggesting that these mutants induce downstream oncogenic signaling without the need to reach the cell surface. Inhibition of constitutive Kit TK activity with dasatinib induced a complex, mature N-glycosylation pattern identical to unstimulated wild-type Kit and resulted in the redistribution of the mutants to the plasma membrane. This relocalization was clearly correlated to the inhibition of TK activity because imatinib, a specific inhibitor of the V560G mutant, inactive on the catalytic D816V mutant, induced only the relocalization of the V560G mutant. These data show that on TK inhibition, the aberrant localization of Kit mutants can be fully reversed. Kit mutants are then exported and/or stabilized at the cell surface as inactive and fully N-glycosylated isoforms.
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Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Benzamidas , Células CHO , Clonación Molecular , Cricetinae , Cricetulus , Dasatinib , Citometría de Flujo , Glicosilación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mesilato de Imatinib , Inmunohistoquímica , Espacio Intracelular/metabolismo , Mutación , Piperazinas/farmacología , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Pirimidinas/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Tiazoles/farmacologíaRESUMEN
Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic acceptor molecules are promising scaffolds for fluorescence resonance energy transfer (FRET)-based nanobiosensors. Over other self-assembling donor-acceptor configurations, our preloaded polymers have the virtue of producing compact assemblies with a fixed donor/acceptor distance. This property, together with the possibility of stoichiometric polymer loading, allowed us to directly address how the FRET efficiency depended on the donor/acceptor. At the population level, nanoprobes based on commercial as well as custom CdSe/ZnS donors displayed the expected dose-dependent rise in transfer efficiency, saturating from about five ATTO dyes/NC. However, for a given acceptor concentration, both the intensity and lifetime of single-pair FRET data revealed a large dispersion of transfer efficiencies, highlighting an important heterogeneity among nominally identical FRET-based nanoprobes. Rigorous quality check during synthesis and shell assembly as well as postsynthesis sorting and purification are required to make hybrid semiconductor-organic nanoprobes a robust and viable alternative to organic or genetically encoded nanobiosensors.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Nanopartículas/química , Nanotecnología/métodos , Ciclohexanos/química , Difusión , Emulsiones , Etanol/química , Hidrólisis , Cinética , Espectroscopía de Resonancia Magnética , Micelas , Microscopía Electrónica de Transmisión/métodos , Modelos Estadísticos , Aceites , TemperaturaRESUMEN
Integration catalyzed by integrase (IN) is a key process in the retrovirus life cycle. Many biochemical or structural human immunodeficiency virus, type 1 (HIV-1) IN studies have been severely impeded by its propensity to aggregate. We characterized a retroviral IN (primate foamy virus (PFV-1)) that displays a solubility profile different from that of HIV-1 IN. Using various techniques, including fluorescence correlation spectroscopy, time-resolved fluorescence anisotropy, and size exclusion chromatography, we identified a monomer-dimer equilibrium for the protein alone, with a half-transition concentration of 20-30 mum. We performed specific enzymatic labeling of PFV-1 IN and measured the fluorescence resonance energy transfer between carboxytetramethylrhodamine-labeled IN and fluorescein-labeled DNA substrates. FRET and fluorescence anisotropy highlight the preferential binding of PFV-1 IN to the 3'-end processing site. Sequence-specific DNA binding was not observed with HIV-1 IN, suggesting that the intrinsic ability of retroviral INs to bind preferentially to the processing site is highly underestimated in the presence of aggregates. IN is in a dimeric state for 3'-processing on short DNA substrates, whereas IN polymerization, mediated by nonspecific contacts at internal DNA positions, occurs on longer DNAs. Additionally, aggregation, mediated by nonspecific IN-IN interactions, occurs preferentially with short DNAs at high IN/DNA ratios. The presence of either higher order complex is detrimental for specific activity. Ionic strength favors catalytically competent over higher order complexes by selectively disrupting nonspecific IN-IN interactions. This counteracting effect was not observed with polymerization. The synergic effect on the selection of specific/competent complexes, obtained by using short DNA substrates under high salt conditions, may have important implications for further structural studies in IN.DNA complexes.
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ADN/química , Integrasas/química , Modelos Químicos , Virus Espumoso de los Simios/enzimología , Proteínas Virales/química , Animales , Catálisis , Transferencia Resonante de Energía de Fluorescencia , Cobayas , Concentración OsmolarRESUMEN
We designed a new efficient and reliable immunosensor and demonstrated its analytic performance to capture breast cancer MCF7 and T47D cells, under laminar flow, onto antibody-coated long alkylsilane self-assembled monolayers (SAMs) in a parallel plate flow chamber. The surface floor of the laminar flow chamber was grafted with an amino-terminated long alkyl chain spacer, 21-aminohenicosyl trichlorosilane (AHTS) followed by tethering a specific monoclonal antibody directed against the human epithelial cell adhesion molecule (EpCAM) antigen, which is overexpressed in primary breast cancer. Properties of the AHTS- and antibody-grafted surface floor were compared to that of surface floors coated with the short alkyl spacers 3-glycidoxy-propyl trimethoxysilane (GPTS) or 3-aminopropyl triethoxysilane (APTES) and antibodies. A theoretical model was constructed according to the geometry of the flow chamber in order to calculate the trajectories that would use cell flows. Cell capture experiments demonstrated that cell immobilization was optimized throughout the whole flow chamber. High cell capture was yielded on antibody-tethered long alkyl AHTS surface. This new procedure offers multiple advantages: a versatile tool readily applied to a panel of purified antibodies, an enrichment of cell immobilization using repetitive cell flow, and a stable capturing surface suitable for long term storage and handling.
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Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos de Neoplasias/inmunología , Técnicas Biosensibles/métodos , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/inmunología , Línea Celular Tumoral , Femenino , Humanos , Silanos/químicaRESUMEN
Sulfur is a functionally important element of living matter. Rhodanese is involved in the enzymatic production of the sulfane sulfur which has been suggested as the biological relevant active sulfur species. Rhodanese domains are ubiquitous structural modules occurring in the three major evolutionary phyla. We characterized a new single-domain rhodanese with a thiosulfate : cyanide transferase activity, Aq-477. Aq-477 can also use tetrathionate and polysulfide. Thermoactivity and thermostability studies show that in solution Aquifex sulfurtranferase exists in equilibrium between monomers, dimers and tetramers, shifting to the tetrameric state in the presence of substrate. We show that oligomerization is important for thermostability and thermoactivity. This is the first characterization of a sulfurtransferase from a hyperthermophilic bacterium, which moreover presents a tetrameric organization. Oligomeric Aq-477 may have been selected in hyperthermophiles because subunit association provides extra stabilization.