Search results for "confocal"

Cytoskeletal stabilization of inhibitory interactions in immunologic synapses of mature human dendritic cells with natural killer cells

2011

Abstract Human mature dendritic cells (DCs) can efficiently stimulate natural killer (NK)–cell responses without being targeted by their cytotoxicity. To understand this important regulatory crosstalk, we characterized the development of the immunologic synapse between mature DCs and resting NK cells. Conjugates between these 2 innate leukocyte populations formed rapidly, persisted for prolonged time periods and matured with DC-derived f-actin polymerization at the synapse. Polarization of IL-12 and IL-12R to the synapse coincided with f-actin polymerization, while other activating and inhibitory molecules were enriched at the interface between DCs and NK cells earlier. Functional assays re…

Novel Paraoxonase 2-Dependent Mechanism Mediating the Biological Effects of the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxo-Dodecanoyl)-l…

2015

ABSTRACT Pseudomonas aeruginosa produces N -(3-oxo-dodecanoyl)- l -homoserine lactone (3OC12), a crucial signaling molecule that elicits diverse biological responses in host cells thought to subvert immune defenses. The mechanism mediating many of these responses remains unknown. The intracellular lactonase paraoxonase 2 (PON2) hydrolyzes and inactivates 3OC12 and is therefore considered a component of host cells that attenuates 3OC12-mediated responses. Here, we demonstrate in cell lines and in primary human bronchial epithelial cells that 3OC12 is rapidly hydrolyzed intracellularly by PON2 to 3OC12 acid, which becomes trapped and accumulates within the cells. Subcellularly, 3OC12 acid acc…

Binding of extracellular matrix proteins to Aspergillus fumigatus conidia

1996

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed…

A practical approach to FRET-based PNA fluorescence in situ hybridization.

2010

Abstract Given the demand for improved methods for detecting and characterizing RNA variants in situ, we developed a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Forster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction as FRET efficiency measure. The FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular …

Ultrastructure of the Interlamellar Membranes of the Nacre of the Bivalve Pteria hirundo, Determined by Immunolabelling.

2015

The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the …

Visualization of Molecular Recognition Events on Microstructured Lipid-Membrane Compartments by In Situ Scanning Force Microscopy This work was suppo…

2002

Fluvastatin stabilizes the blood–brain barrier in vitro by nitric oxide-dependent dephosphorylation of myosin light chains

2006

Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme-A reductase and the downstream mevalonate pathway is in part responsible for the beneficial effects that statins exert on the cardiovascular system. In this study we aimed at analysing the stabilizing effects of fluvastatin on the blood-brain barrier (BBB) integrity, using an in vitro co-culture model of ECV304 and C6, or primary bovine endothelial cells and rat astrocytes. Fluvastatin dose-dependently (1-25 micromol/l) increased barrier integrity as analysed by measurements of transendothelial electrical resistance (TEER). This effect (117.4+/-2.6% at 25 micromol/l) was significantly reduced by the nitric oxide (NO) synthase inhibitor L…

Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system

2005

Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In …

Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel α2β1 Integrin Internalization Pathway

2013

We have previously shown that soluble collagen and a human pathogen, echovirus 1 (EV1) cluster α2β1 integrin on the plasma membrane and cause their internalization into cytoplasmic endosomes. Here we show that cholesterol plays a major role not only in the uptake of α2β1 integrin and its ligands but also in the formation of α2 integrin-specific multivesicular bodies (α2-MVBs) and virus infection. EV1 infection and α2β1 integrin internalization were totally halted by low amounts of the cholesterol-aggregating drugs filipin or nystatin. Inhibition of cholesterol synthesis and accumulation of lanosterol after ketoconazole treatment inhibited uptake of collagen, virus and clustered integrin, an…

Internalization of Echovirus 1 in Caveolae

2002

ABSTRACT Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor α2β1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, α2β1 integrin, and …