Gold-substituted Silver-intensified Peroxidase Immunolabeling for FIB-SEM Imaging.

Modern electron microscopy offers a wide variety of tools to investigate the ultrastructural organization of cells and tissues and to accurately pinpoint intracellular localizations of macromolecules of interest. New volumetric electron microscopy techniques and new instrumentation provide unique opportunities for high-throughput analysis of comparatively large volumes of tissue and their complete reconstitution in three-dimensional (3D) electron microscopy. However, due to a variety of technical issues such as the limited penetration of label into the tissue, low antigen preservation, substantial electron density of secondary detection reagents, and many others, the adaptation of immuno-detection techniques for use with such 3D imaging methods as focused ion beam-scanning electron microscopy (FIB-SEM) has been challenging.

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells.

Background: Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods: We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Corticotropin-releasing factor and urocortin regulate spine and synapse formation: structural basis for stress-induced neuronal remodeling and pathology.

Dendritic spines are important sites of excitatory neurotransmission in the brain with their function determined by their structure and molecular content. Alterations in spine number, morphology and receptor content are a hallmark of many psychiatric disorders, most notably those because of stress. We investigated the role of corticotropin-releasing factor (CRF) stress peptides on the plasticity of spines in the cerebellum, a structure implicated in a host of mental illnesses, particularly of a developmental origin.

Surface characteristics of nanoparticles determine their intracellular fate in and processing by human blood-brain barrier endothelial cells in vitro.

A polarized layer of endothelial cells that comprises the blood-brain barrier (BBB) precludes access of systemically administered medicines to brain tissue. Consequently, there is a need for drug delivery vehicles that mediate transendothelial transport of such medicines. Endothelial cells use a variety of endocytotic pathways for the internalization of exogenous materials, including clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis.

Formation of E-cadherin/beta-catenin-based adherens junctions in hepatocytes requires serine-10 in p27(Kip1).

The adhesion between epithelial cells at adherens junctions is regulated by signaling pathways that mediate the intracellular trafficking and assembly of its core components. Insight into the molecular mechanisms of this is necessary to understand how adherens junctions contribute to the functional organization of epithelial tissues. Here, we demonstrate that in human hepatic HepG2 cells, oncostatin M-p42/44 mitogen-activated protein kinase signaling stimulates the phosphorylation of p27(Kip1) on Ser-10 and promotes cell-cell adhesion.

Adhesion receptors mediate efficient non-viral gene delivery.

For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors, non-viral vectors pose minimal health risks, but to meet therapeutic requirements their efficacy needs major improvement. This goal may be accomplished by better defining the mechanism of non-viral gene delivery and exploiting specific cellular properties.

The dynamic developmental localization of the full-length corticotropin-releasing factor receptor type 2 in rat cerebellum.

Corticotropin releasing factor receptor 2 (CRF-R2) is strongly expressed in the cerebellum and plays an important role in the development of the cerebellar circuitry, particularly in the development of the dendritic trees and afferent input to Purkinje cells. However, the mechanisms responsible for the distribution and stabilization of CRF-R2 in the cerebellum are not well understood. Here, we provide the first detailed analysis of the cellular localization of the full-length form of CRF-R2 in rat cerebellum during early postnatal development.

Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells.

The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis.

CRF and urocortin differentially modulate GluRdelta2 expression and distribution in parallel fiber-Purkinje cell synapses.

Corticotropin-releasing factor (CRF) and urocortin (UCN) are closely related multifunctional regulators, governing, among other processes, Purkinje cell development. Here, we investigate the effects of CRF and UCN on Purkinje cells in organotypic slices. We show that both peptides upregulate delta2 ionotropic glutamate receptor gene expression, and increase the abundance of the receptor in the postsynaptic density.

Cilia in the head of hornets: form and function.

In the head of the Oriental hornet, beneath the cuticle, there are plaques of hair cells. These are distributed throughout the upper front part of the head; to wit: in the region of the vertex (i.e.

Ontogenesis of peripheral electromagnetic receptors in hornets.

This article traces the ontogenesis of peripheral electromagnetic receptors (PER) in the cuticle of the Oriental hornet (Vespa orientalis). In the abdominal cuticle of adult hornets, the PERs are densely distributed throughout, but there are even more than 30 at the margins of the segments. These organelles develop as a network in the hornet cuticle immediately upon its completion.

The postnatal developmental expression pattern of urocortin in the rat olivocerebellar system.

Urocortin belongs to the family of corticotropin-releasing factor (CRF)-like peptides, which play an important role in sensorimotor coordination. CRF induces locomotor activity, and urocortin has an inhibitory effect. Here, we document the regional and subcellular localization of urocortin in the developing rat cerebellum to compare it with CRF.

Corticotropin-releasing factor receptor types 1 and 2 are differentially expressed in pre- and post-synaptic elements in the post-natal developing rat cerebellum.

Corticotropin-releasing factor (CRF)-like proteins act via two G-protein-coupled receptors (CRF-R1 and CRF-R2) playing important neuromodulatory roles in stress responses and synaptic plasticity. The cerebellar expression of corticotropin-releasing factor-like ligands has been well documented, but their receptor localization has not. This is the first combination of a light microscopic and ultrastructural study to localize corticotropin-releasing factor receptors immunohistologically in the developing rat cerebellum.

AFM study of microstructures on the cornea of the compound eye and ocelli of the hornet Vespa orientalis (Insecta, Hymenoptera).

The present study describes a novel structure occurring on the cornea in the compound eye and the ocellus of the Oriental hornet. This description is based on observations carried out via scanning electron microscopy (SEM) and primarily via atomic force microscopy (AFM). We report herein that the vespan cornea is densely covered with cupola-shaped protrusions, which in the compound eye have bases about 0.

The localisation of urocortin in the adult rat cerebellum: a light and electron microscopic study.

Light and electron microscopic immunocytochemistry was used to identify the cellular and subcellular localisation of urocortin in the adult rat cerebellum. Urocortin immunoreactivity (UCN-ir) was visualised throughout the cerebellum, yet predominated in the posterior vermal lobules, especially lobules IX and X, the flocculus, paraflocculus and deep cerebellar nuclei. Cortical immunoreactivity was most evident in the Purkinje cell layer and molecular layer.

Are ciliary hair cells and photoreceptors components of a gravitic system of the hornet Vespa orientalis?

Social wasps, including the sub-family Vespinae, are social insects that build combs beneath the ground which are directed towards the gravitic pull of the earth, and this in dim light or complete darkness. On the inner side of the frons plate in social wasps there is a gravity sensing apparatus composed of static and dynamic nerve fibers, some of which connect between the frons plate and the brain. It is highly probable that the interaction between the fibers and the various structures in the head is responsible for the proprioceptive ability of hornets, including gravity detection.

Micromorphology of the dorsal ocelli of the Oriental hornet.

The development of vision in animals throughout evolution has been reviewed by Sir Stewart Duke-Elder, whose survey of the sense of sight ranges from lowly Crustaceans to mammals and man. According to Duke-Elder each ocellus is formed by the "fusion of two or more ocelli, each with its own retina and pigment cup". This process of 'ocellation' probably occurred independently in a number of phyla.

Nonpenetrating vascular clips for small-caliber anastomosis.

In the search for better anastomosing techniques, an improved vascular stapler device (VCS clip applier system(R)) has been introduced. The system uses nonpenetrating clips to approximate everted vessel walls. The objective of this study was to determine the effects of nonpenetrating vascular clips on endothelial wound healing.

Lens epithelial cell layer formation related to hydrogel foldable intraocular lenses.

Purpose: To determine the incidence of postoperative lens epithelial cell (LEC) layer formation anterior to the Hydroview hydrogel foldable intraocular lens (IOL), the effect on vision, and the appearance under scanning electron microscopy (SEM).

Setting: Eye Unit of a district general hospital, Delfzijl, and the Laboratory for Cell Biology and Electron Microscopy, Faculty of Medical Sciences, University of Groningen, The Netherlands.

Methods: In the prospective study, 207 eyes that received a Hydroview hydrogel foldable IOL during 1996 were followed for almost 2 years (range 8 to 98 weeks).

Non-coating fixation techniques or redundancy of conductive coating, low kV FE-SEM operation and combined SEM/TEM of biological tissues.

Non-coating fixation methods, in particular the tannic acid/arginine/osmium tetroxide procedure, are employed for a number of reasons on the guinea-pig organ of Corti hair cell stereocilia glycocalyx and the imprints of the stereocilia at the bottom side of the tectorial membrane, and on the rat and cat intestinal epithelial microvilli glycocalyx and mucus-producing goblet cells. These methods are used firstly to confirm that non-coating prepared specimens can be embedded for TEM observation at 60-100 kV without loss of detail information, and these images can be compared with cryo-FE-SEM images of the same structure/tissue. Secondly, they show that specimens treated according non-coating techniques become optimally preserved and electrically conductive, so that no external conductive coating is required.

Extralysosomal localisation of acid phosphatase in the rat kidney.

There is strong evidence that acid phosphatase (AcPase) plays an important role in the catabolism of the glomerular basement membrane (GBM) and the removal of macromolecular debris resulting from ultrafiltration. Recent enzyme histochemical investigations provide new evidence of the antithrombotic and anti-inflammatory function of ADPase and on the distribution of AcPase in mouse kidney tubule cells. By means of 3 mM cerium as the trapping agent and 1 mM p-nitrophenyl phosphate as the substrate, extralysosomal AcPase could be demonstrated at the ultrastructural level.

A comparative study of thin coatings of Au/Pd, Pt and Cr produced by magnetron sputtering for FE-SEM.

Visualization of structural details of specimens in field emission scanning electron microscopy (FE-SEM) requires optimal conductivity. This paper reports on the differences in conductive layers of Au/Pd, Pt and Cr, with a thickness of 1.5–3.

Demonstration of the fine structure of stereocilia in the organ of Corti of the guinea pig by field emission scanning electron microscopy.

A combined perfusion- and immersion prefixation with glutaraldehyde followed by a tannic acid/arginine/osmium tetroxide (TAO) treatment of the guinea pig cochlea is described for field-emission gun scanning electron microscopy (FEG-SEM) observation of the fine structure of the stereocilia of the organ of Corti. Conventional osmium tetroxide postfixation methods in combination with a thin conductive coating failed to show the fine structure of the glycocalyx of the epithelial lining in the endolymphatic compartment of the cochlea, in particular, on the stereocilia surface. The antennulae-like glycocalyx covering of the stereocilia surface of the more pronounced rows of outer hair cells has been demonstrated only in ultrathin sections by means of cationic markers.

Low-voltage field-emission scanning electron microscopy of non-coated guinea-pig hair cell stereocilia.

The stereociliar structures of the guinea-pig cochlear organ of Corti were studied at low-voltage (1-5 kV) with field-emission scanning electron microscope (SEM) using various pre- and post-fixation methods, such as OTOTO (OsO4/thiocrbohydrazide/OsO4/thiocarbohydrazide/OsO4) and TAO (tannic acid/arginine/OsO4), and different dissection procedures of the cochlea. A perfusion and immersion pre-fixation with glutaraldehyde, in combination with removal of the bony wall and stria vascularis from the cochlea, followed by the TAO non-coating treatment gave the best result at 2 kV acceleration voltage. Due to these new technique, several interesting delicate structures of the stereocilia, in particular fine surface structures, were detected for the first time using SEM.