BackgroundCigarette 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.MethodsWe 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.ResultsWe observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.ConclusionThe observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.

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. The different modes of endocytosis result in the delivery of endocytosed material to distinctive intracellular compartments and therewith correlated differential processing. To obtain insight into the properties of drug delivery vehicles that direct their intracellular processing in brain endothelial cells, we investigated the intracellular processing of fixed-size nanoparticles in an in vitro BBB model as a function of distinct nanoparticle surface modifications. Caveolar endocytosis, adsorptive-mediated endocytosis, and receptor-mediated endocytosis were promoted by the use of uncoated 500-nm particles, attachment of the cationic polymer polyethyleneimine (PEI), and attachment of prion proteins, respectively. We demonstrate that surface modifications of nanoparticles, including charge and protein ligands, affect their mode of internalization by brain endothelial cells and thereby their subcellular fate and transcytotic potential.

The structure of the chorion and plasma membranes of gastrula‐stage zebrafish Brachydanio rerio embryos were studied using field emission scanning electron microscopy (FE‐SEM) and transmission electron microscopy (TEM). These studies confirm the outer chorion membrane complex to be 1.5–2.5 μm in thickness and to consist of three layers, electron‐dense outer and innermost layers (0.2–0.3 and 1.0–1.6 μm in thickness respectively) separated by an electron‐lucent middle layer (0.3–0.6 μm in thickness). The middle and inner layers are pierced by pore canals. A granular to farinaceous nature of the thin outer surface of the outer layer of the chorion has been revealed for the first time. The study provides original TEM images of the plasma membrane structures of gastrula‐stage embryos, and FE‐SEM and TEM images showing the plasma membrane to have three morpohologically distinct regions, being prominently ridged and folded at the surface of the blastoderm, smooth over the syncytial layer at the vegetal pole and with an intermediate region between the animal and vegetal pole where folding develops in advance of the expanding blastodermal disc of cells. FE‐SEM and TEM studies reveal details of the syncytial layer (1–4 μm thick) beneath the smooth plasma membrane at the vegetal pole, containing cytoplasmic organelles and small yolk globules. The significance of the structural detail shown in these studies is considered in the light of the difficulties experienced in cryopreservation of the embryo resulting from the inability of achieving cryoprotectant penetration of the yolk mass.

Using a mutant hepatocyte cell line in which E-cadherin and ␤-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/␤-cateninbased adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/␤-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.

Strong cationic polyethyleneimine (PEI) is used to trace anionic sites in lung, choroid plexus, glomeruli, blood vessels and also on collagen fibrils. In the mentioned tissues, PEI is found in the basement membranes with a spacing identical to that found on collagen fibrils. It is assumed that the acid endgroups of tropocollagen molecules are responsible for the binding of PEI.

In this study the morphologic and functional changes were compared after irradiation (single dose, 15 Gy) of rat submandibular salivary glands. Before and 1-10 days after local irradiation of the gland region, samples of submandibular saliva were collected after stimulation by pilocarpine. At the same time-points and also 3 h postirradiation submandibular glands were carefully extirpated and prepared for histocytologic examination (LM, TEM). Maximal increase of the lag phase and decrease of the flow rate were observed 3 days after irradiation, while [K+] and [Na+] increased and decreased, respectively, from days 1 and 3 after irradiation. Morphologic changes were observed from the third hour after irradiation, were maximal 3 days after irradiation and had partially recovered by day 10. Three hours and 1 day after irradiation degranulation of convoluted granulated tubes (CGT) was observed. Three days after irradiation the most striking morphologic changes in serous and mucous cells were distension of the cisternae of the RER, degeneration of mitochondria and vacuolization of the cytoplasm. Fibril-like condensations of electron dense material in the mucous granules were observed 3 h, 1 and 6 days after irradiation. Regranulation of CGT cells was observed from day 6. From this study it is concluded that changes in salivary gland function can be observed before major morphologic changes occur. Functional changes persist after the morphologic changes seem to have virtually returned to normal.

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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. Both CRF-R1 and CRF-R2 were expressed in climbing fibres from early stages (post-natal day 3) to the adult, but CRF-R2 immunoreactivity was only prominent throughout the molecular layer in the posterior cerebellar lobules. CRF-R1 immunoreactivity was concentrated in apical regions of Purkinje cell somata and later in primary dendrites exhibiting a diffuse cytoplasmic appearance. In Purkinje cells, CRF-R1 immunoreactivity was never membrane bound post-synaptically in dendritic spines while CRF-R2 immunoreactivity was found on plasmic membranes of Purkinje cells from post-natal day 15 onwards. We conclude that the localization of these receptors in cerebellar afferents implies their pre-synaptic control of the release of corticotropin-releasing factor-like ligands, impacting on the sensory information being transmitted from afferents. Furthermore, the fact that CRF-R2 is membrane bound at synapses, while CRF-R1 is not, suggests that ligands couple to CRF-R2 via synaptic transmission and to CRF-R1 via volume transmission. Finally, the distinct expression profiles of receptors along structural domains of Purkinje cells suggest that the role for these receptors is to modulate afferent inputs.