Quasi-periodic substructure in the microvessel endothelial glycocalyx: a possible explanation for molecular filtering?

The luminal surface of endothelial cells is lined with the glycocalyx, a network structure of glycoproteins probably 50 to 100 nm thick. It has been suggested that a relatively regular fibre-matrix structure may be responsible for the ultrafiltration properties of microvascular walls, both when the endothelium is continuous and when it is fenestrated. Positive structural evidence demonstrating an underlying periodicity in the glycocalyx has been hard to obtain. Here we present structural analysis of glycocalyx samples prepared in a variety of ways for electron microscopy. Using computed autocorrelation functions and Fourier transforms of representative areas of the electron micrograph images, we show that there is an underlying three-dimensional fibrous meshwork within the glycocalyx with characteristic spacings of about 20 nm. Together with a fibre diameter consistent with our observations of about 10-12 nm, the 20-nm spacing provides just the size regime to account satisfactorily for the observed molecular filtering; the observations are consistent with the fibre matrix model. We also show that the fibrous elements may occur in clusters with a common intercluster spacing of about 100 nm and speculate that this may reveal organisation of the glycocalyx by a quasi-regular submembranous cytoskeletal scaffold.     

Bacterial biofilms and surface fouling

Bacteria are attracted to surfaces. Their surface adhesion, with subsequent binary fission and exopolymer production, leads to the formation of biofilms. Such biofilms consist of bacterial cells in a matrix of their own exopolysaccharide glycocalyces. In addition to the bulk fluid and the surface, biofilms constitute a third physical phase. The close proximity of the bacterial cells in the biofilm matrices assists the formation of metabolically dependent consortia. The chemical and physical activities of these microbial communities produces a heterogeneous system at the colonised surface. Metabolites, produced at specific points on the surface, can lead to the development of effective anodes and cathodes at adjoining locations on the surface. In this way the fouling of a surface by bacterial biofilm development facilitates focal attack on that surface. This pit formation is characteristic of bacterial surface activities as diverse as dental decay and metal corrosion. In this review, we examine bacterial adhesion, biofilm formation and several instances of focal bacterial attack on colonised surfaces. However, pathogenic biofilms and the fouling of biological surfaces, with the exception of caries formation, is outside the scope of this paper.     

Large-Conductance Ca2+ -Activated K+ Channels in Freshly Dissociated Smooth Muscle Cells

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca²⁺-activated K⁺ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca²⁺-activated, TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca²⁺-activated K⁺ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.     

The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor-mediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K+ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-beta-cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, alpha(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes alpha(2,3)-, but not with Sambucus nigra agglutinin that recognizes alpha(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal alpha(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.     

Development of Pollen Grain Walls and Accumulation of Sporopollenin

By means of electron microscopy, we studied the development of pollen grain walls in Calendula officinalis L., Dimorphotheca aurantiaca DC., and Cichorium intybus L. (Asteraceae). As a reference, we studied the plants from the families Schisandraceae (Schisandra chinensis (Turcz.) Baill.), Lauraceae (Persea americana Mill.), Boraginaceae (Borago officinalis L.), and Cycadaceae (Encephalartos altensteinii Lehm.). In Asteraceae, we revealed two successively initiated layers of glycocalyx that form outer and inner layers of the ectexine. The formation of endexine is contributed by plasma membrane and small vesicles. Glycocalyx in the plants from the families Schisandraceae, Lauraceae, Boraginaceae, and Cycadaceae was found to consist of radially arranged helical cylindrical units, which are receptors of sporopollenin deposition. It is assumed that the receptor-independent accumulation of sporopollenin is also possible.     

Biofilms in device-related infections

The use of various medical devices including indwelling vascular catheters, cardiac pacemakers, prosthetic heart valves, chronic ambulatory peritoneal dialysis catheters and prosthetic joints has greatly facilitated the management of serious medical and surgical illness. However, the successful development of synthetic materials and introduction of these artificial devices into various body systems has been accompanied by the ability of microorganisms to adhere to these devices in the environment of biofilms that protect them from the activity of antimicrobial agents and from host defense mechanisms. A number of host, biomaterial and microbial factors are unique to the initiation, persistence and treatment failures of device-related infections. Intravascular catheters are the most common devices used in clinical practice and interactions associated with these devices are the leading cause of nosocomial bacteremias. The infections associated with these devices include insertion site infection, septic thrombophlebitis, septicemia, endocarditis and metastatic abscesses. Other important device-related infections include infections of vascular prostheses, intracardiac prostheses, total artificial hearts, indwelling urinary catheters, orthopedic prostheses, endotracheal tubes and extended wear lenses. The diagnosis and management of biofilm-associated infections remain difficult but critical issues. Appropriate antimicrobial therapy is often not effective in eradicating these infections and the removal of the device becomes necessary. Several improved diagnostic and therapeutic modalities have been reported in recent experimental studies. The clinical usefulness of these strategies remains to be determined.     

IL-22 promotes the formation of a MUC17 glycocalyx barrier in the postnatal small intestine during weaning

1. Download : Download high-res image (162KB)
2. Download : Download full-size image

     

Structural alterations of the human erythrocyte membrane upon influenza virus attachment

Molecular events on the human erythrocyte membrane subsequent to influence virus binding were investigated by electron spin resonance (ESR) measurements after spin labeling of the cell membrane at different positions. Virus binding affected the glycocalyx structure as well as the physical state of the cytoskeleton at the inner leaflet, but not the lipid phase. A lateral reorganization of spin-labeled glycophorin was not indicated after virus attachment.     

Fertilization cones of inseminated sea urchin (Arbacia punctulata) oocytes: Development of an asymmetry in plasma membrane topography

Electron microscopic and cytochemical investigations were carried out on inseminated Arbacia oocytes comparing structural and chemical properties of their microvillous surface and fertilization cones. Early fertilization cones (up to 4 min postinsemination) were relatively small, smooth surface projections of cytoplasm that engulfed the incorporated sperm nucleus. However, in contrast to surrounding microvillous areas of the oocyte surface, enlarged fertilization cones (8 to 15 min postinsemination) had a distinctive crenated appearance that persisted until their regression. When examined by various cytochemical techniques, membrane delimiting fertilization cones had a much lower affinity for agents that stain negatively charged and carbohydrate moieties (cationized ferritin, concanavalin A, ruthenium red, and alcian blue) than did other regions of the oocyte surface. This difference in surface properties of membrane delimiting the site of sperm-egg fusion was not due solely to incorporated sperm plasma membrane and did not occur when inseminated oocytes were incubated with cytochalasin B. Little or no difference in the membrane of the fertilization cone versus microvillous areas was observed when inseminated eggs were freeze-fractured or prepared with agents (filipin and polymixin B) to demonstrate β-hydroxysterols and anionic phospholipids. These observations indicate that membrane delimiting the fertilization cone differs from the remainder of the oocyte surface and suggests that following insemination significant rearrangements of surface molecules take place within the egg plasmalemma that give rise to asymmetries in membrane topography.