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1.
Am J Pathol ; 191(1): 194-203, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33069718

RESUMEN

Contraction of vascular smooth muscle is regulated primarily by calcium concentration and secondarily by ROCK activity within the cells. In contrast to the wealth of information regarding regulation of calcium concentration, little is known about the spatiotemporal regulation of ROCK activity in live blood vessels. Here, we report ROCK activation in subcutaneous arterioles in a transgenic mouse line that expresses a genetically encoded ROCK biosensor based on the principle of FÓ§rster resonance energy transfer by two-photon excitation in vivo imaging. Rapid vasospasm was induced upon laser ablation of arterioles, concomitant with a transient increase in calcium concentration in arteriolar smooth muscles. Unlike the increase in calcium concentration, vasoconstriction and ROCK activation continued for several minutes after irradiation. Both the ROCK inhibitor, fasudil, and the ganglionic nicotinic acetylcholine receptor blocker, hexamethonium, inhibited laser-induced ROCK activation and reduced the duration of vasospasm at the segments distant from the irradiated point. These observations suggest that vasoconstriction is initially triggered by a rapid surge of cytoplasmic calcium and then maintained by sympathetic nerve-mediated ROCK activation.


Asunto(s)
Músculo Liso Vascular/enzimología , Vasoconstricción/fisiología , Quinasas Asociadas a rho/metabolismo , Animales , Sistema Nervioso Autónomo/fisiología , Señalización del Calcio/fisiología , Transferencia Resonante de Energía de Fluorescencia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Liso Vascular/inervación
2.
Pharm Res ; 37(12): 248, 2020 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-33230672

RESUMEN

PURPOSE: We have previously reported that Capryol 90 improves the intestinal absorption of insulin, a peptide drug, without causing serious damage to the intestinal epithelium. However, the effects of Capryol 90 and its related formulations on the intestinal absorption of other drugs, and their absorption-enhancing mechanisms are still unclear. The aim of this study is to evaluate the effects of Capryol 90 and its related formulations on the intestinal absorption of drugs and elucidate their absorption-enhancing mechanisms. METHODS: The intestinal absorption of 5(6)-carboxyfluorescein, fluorescein isothiocyanate-dextrans, and alendronate was evaluated using an in situ closed loop method. Brush border membrane vesicles (BBMVs) were labeled with fluorescent probes, and the fluidity of membrane was evaluated by a fluorescence depolarization method. The expression levels of tight junction (TJ) proteins were measured using a Western blot method and immunofluorescence staining. RESULTS: Among the tested excipients, Capryol 90 significantly improved the small and large intestinal absorption of drugs. In mechanistic studies, Capryol 90 increased the membrane fluidity of lipid bilayers in BBMVs. Additionally, Capryol 90 decreased the expression levels of TJ-associated proteins, namely claudin-4, occludin, and ZO-1. CONCLUSIONS: Capryol 90 is an effective absorption enhancer for improving the intestinal absorption of poorly absorbed drugs via both transcellular and paracellular pathways.


Asunto(s)
Alendronato/metabolismo , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Polímeros/farmacología , Glicoles de Propileno/farmacología , Animales , Células CACO-2 , Claudina-4/metabolismo , Dextranos/metabolismo , Impedancia Eléctrica , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceínas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Masculino , Fluidez de la Membrana/efectos de los fármacos , Ocludina/metabolismo , Ratas Wistar , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo
3.
Cell Struct Funct ; 44(2): 153-169, 2019 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-30905922

RESUMEN

Two decades have passed since the development of the first calcium indicator based on the green fluorescent protein (GFP) and the principle of Förster resonance energy transfer (FRET). During this period, researchers have advanced many novel ideas for the improvement of such genetically encoded FRET biosensors, which have allowed them to expand their targets from small molecules to signaling proteins and physicochemical properties. Although the merits of "genetically encoded" FRET biosensors became clear once various cell lines were established and several transgenic organisms were generated, the road to these developments was not necessarily a smooth one. Moreover, even today the development of new FRET biosensors remains a very labor-intensive, trial-and-error process. Therefore, at this junction, it may be worthwhile to summarize the progress of the FRET biosensor and discuss the future direction of its development and application.Key words: FRET, biosensor, fluorescent protein.


Asunto(s)
Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Organismos Modificados Genéticamente/genética , Animales , Humanos
4.
Cell Struct Funct ; 43(2): 129-140, 2018 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-29962383

RESUMEN

For more than a century, hematoxylin and eosin (H&E) staining has been the de facto standard for histological studies. Consequently, the legacy of histological knowledge is largely based on H&E staining. Due to the recent advent of multi-photon excitation microscopy, the observation of live tissue is increasingly being used in many research fields. Adoption of this technique has been further accelerated by the development of genetically encoded biosensors for ions and signaling molecules. However, H&E-based histology has not yet begun to fully utilize in vivo imaging due to the lack of proper morphological markers. Here, we report a genetically encoded fluorescent marker, NuCyM (Nucleus, Cytosol, and Membrane), which is designed to recapitulate H&E staining patterns in vivo. We generated a transgenic mouse line ubiquitously expressing NuCyM by using a ROSA26 bacterial artificial chromosome (BAC) clone. NuCyM evenly marked the plasma membrane, cytoplasm and nucleus in most tissues, yielding H&E staining-like images. In the NuCyM-expressing cells, cell division of a single cell was clearly observed as five basic phases during M phase by three-dimensional imaging. We next crossed NuCyM mice with transgenic mice expressing an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). Using NuCyM, ERK activity in each cell could be extracted from the FRET images. To further accelerate the image analysis, we employed machine learning-based segmentation methods, and thereby automatically quantitated ERK activity in each cell. In conclusion, NuCyM is a versatile cell morphological marker that enables us to grasp histological information as with H&E staining.Key words: in vivo imaging, histology, machine learning, molecular activity.


Asunto(s)
Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Imagenología Tridimensional/métodos , Sistema de Señalización de MAP Quinasas , Aprendizaje Automático , Análisis de la Célula Individual/métodos , Animales , Perros , Células de Riñón Canino Madin Darby , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente/métodos
5.
Cell Struct Funct ; 42(1): 1-13, 2017 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-27885213

RESUMEN

Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.


Asunto(s)
Técnicas Biosensibles , Quinasas Asociadas a rho/metabolismo , Secuencia de Aminoácidos , Western Blotting , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Microscopía Fluorescente , Fosforilación , Plásmidos/genética , Plásmidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especificidad por Sustrato , Imagen de Lapso de Tiempo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genética
6.
Sci Rep ; 8(1): 8984, 2018 06 12.
Artículo en Inglés | MEDLINE | ID: mdl-29895862

RESUMEN

Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.

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