Intracellular localization of factor VIII-related antigen and fibronectin in cultured human endothelial cells: evidence for divergent routes of intracellular translocation

Cultured human endothelial cells synthesize and secrete both fibronectin and factor VIII-related antigen (VIIIR:Ag). In immunofluorescence microscopy, intracellular fibronectin was seen diffusely perinuclearly whereas VIIIR:Ag was located both diffusely in the perinuclear cytoplasm and in distinct rod-shaped granules. These granules could, moreover, be visualized with fluorochrome-coupled Ricinus communis agglutinin I (RCA), which also stained the Golgi apparatus as a reticular juxtanuclear structure, and they were identified as Weibel-Palade bodies by immunoelectron microscopy. Puromycin treatment depleted intracellular fibronectin but did not affect the granular localization of VIIIR:Ag. A short exposure of the cells to monensin caused a juxtanuclear accumulation of fibronectin at the Golgi region whereas VIIIR:Ag only was seen in rounded cytoplasmic granules. A prolonged monensin treatment brought about a cytoplasmic accumulation of fibronectin-containing vesicles whereas VIIIR:Ag showed no accumulation and there was no codistribution between granules containing fibronectin or VIIIR:Ag. Type IV procollagen, on the other hand, was distinctly co-localized with fibronectin. In monensin-treated cells RCA mainly stained the VIIIR:Ag-containing vesicles whereas Concanavalin A (Con A) appeared to label the fibronectin-containing vesicles. Immunoelectron microscopy of these cells revealed VIIIR:Ag in some vacuolar structures and typical Weibel-Palade bodies could not be identified. Exposure of the cells to tunicamycin, on the other hand, caused a prominent cytoplasmic accumulation of VIIIR:Ag and, within 96 h, led to the disappearance of most of the VIIIR:Ag-positive granules but did not affect the intracellular distribution of fibronectin. These results, which show that metabolical inhibitors affect differently the intracellular compartmentalization of fibronectin and VIIIR:Ag, indicate, that the two glycoproteins have divergent intracellular pathways in cultured human endothelial cells.     

Differential Regulation and Function of CD73, a Glycosyl-Phosphatidylinositol–linked 70-kD Adhesion Molecule, on Lymphocytes and Endothelial Cells

CD73, otherwise known as ecto-5′-nucleotidase, is a glycosyl-phosphatidylinositol–linked 70-kD molecule expressed on different cell types, including vascular endothelial cells (EC) and certain subtypes of lymphocytes. There is strong evidence for lymphocyte CD73 having a role in several immunological phenomena such as lymphocyte activation, proliferation, and adhesion to endothelium, but the physiological role of CD73 in other cell types is less clear. To compare the biological characteristics of CD73 in different cell types, we have studied the structure, function, and surface modulation of CD73 on lymphocytes and EC. CD73 molecules on lymphocytes are shed from the cell surface as a consequence of triggering with an antiCD73 mAb, mimicking ligand binding. In contrast, triggering of endothelial CD73 does not have any effect on its expression. Lymphocyte CD73 is susceptible to phosphatidylinositol phospholipase, whereas only a small portion of CD73 on EC could be removed by this enzyme. Furthermore, CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon mAb triggering, whereas triggering of lymphocyte CD73 can induce tyrosine phosphorylation. Despite the functional differences, CD73 molecules on lymphocytes and EC were practically identical structurally, when studied at the protein, mRNA, and cDNA level. Thus, CD73 is an interesting example of a molecule which lacks structural variants but yet has a wide diversity of biological functions. We suggest that the ligand- induced shedding of lymphocyte CD73 represents an important and novel means of controlling lymphocyte– EC interactions.     

Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of tobacco mosaic virus

The flavum strain of Tobacco mosaic virus (TMV) differs from the wild-type (wt) virus by causing strong yellow and green mosaic in the systemically infected developing leaves, yellowing in the fully expanded leaves, and distinct malformations of chloroplasts in both types of infected tissues. Analysis of the thylakoid proteins of flavum strain-infected tobacco leaves indicated that the chlorosis in mature leaves was accompanied by depletion of the entire photosystem II (PSII) core complexes and the 33-kDa protein of the oxygen evolving complex. The only change observed in the thylakoid proteins of the corresponding wt TMV-infected leaves was a slight reduction of the alpha and beta subunits of the ATP synthase complex. The coat proteins of different yellowing strains of TMV are known to effectively accumulate inside chloroplasts, but in this work, the viral movement protein also was detected in association with the thylakoid membranes of flavum strain-infected leaves. The mRNAs of different enzymes involved in the chlorophyll biosynthesis pathway were not reduced in the mature chlorotic leaves. These results suggest that the chlorosis was not caused by reduction of pigment biosynthesis, but rather, by reduction of specific proteins of the PSII core complexes and by consequent break-down of the pigments.     

Decomposition and element concentrations of Norway spruce needle litter with differing B, N, or P status

Adequate boron (B) nutrition may decrease concentrations of phenolic compounds and enhance structural integrity and lignification in plants, compared with suboptimal B. This could affect decomposition in areas where B deficiencies are common. The mass loss and changes in element concentrations in Norway spruce needle litter were studied with combinations of litter from high-B and low-B trees, incubated for 29 months, in either B fertilised or control plots without B addition. The litter originated from the same Norway spruce field experiments. Additionally, the field experiments included long-term N and P treatments. Initially, lowest lignin concentrations were found in Norway spruce litter from the treatment P and particularly in the combination B?+?P, and highest in the B?+?N fertilised plots. The mass loss of Norway spruce litter was not affected by the treatments. However, B_litter increased Cu accumulation. The litter from the B?+?P fertilised plots accumulated considerably more Al, Ca, S and Zn than the other treatments, whereas B together with N reduced the remaining amounts of these elements. Reduced nutrient release from litter may have far-reaching consequences on nutrient cycles in forests.     

Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.     

Physiological and autonomic stress responses after prolonged sleep restriction and subsequent recovery sleep in healthy young men

Sleep and Biological Rhythms - Sleep restriction is increasingly common and associated with the development of health problems. We investigated how the neuroendocrine stress systems respond to...     

Formation of vinculin plaques precedes other cytoskeletal changes during retinoic acid-induced teratocarcinoma cell differentiation

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.     

Co-Exposure with Fullerene May Strengthen Health Effects of Organic Industrial Chemicals

In vitro toxicological studies together with atomistic molecular dynamics simulations show that occupational co-exposure with C₆₀ fullerene may strengthen the health effects of organic industrial chemicals. The chemicals studied are acetophenone, benzaldehyde, benzyl alcohol, m-cresol, and toluene which can be used with fullerene as reagents or solvents in industrial processes. Potential co-exposure scenarios include a fullerene dust and organic chemical vapor, or a fullerene solution aerosolized in workplace air. Unfiltered and filtered mixtures of C₆₀ and organic chemicals represent different co-exposure scenarios in in vitro studies where acute cytotoxicity and immunotoxicity of C₆₀ and organic chemicals are tested together and alone by using human THP-1-derived macrophages. Statistically significant co-effects are observed for an unfiltered mixture of benzaldehyde and C₆₀ that is more cytotoxic than benzaldehyde alone, and for a filtered mixture of m-cresol and C₆₀ that is slightly less cytotoxic than m-cresol. Hydrophobicity of chemicals correlates with co-effects when secretion of pro-inflammatory cytokines IL-1β and TNF-α is considered. Complementary atomistic molecular dynamics simulations reveal that C₆₀ co-aggregates with all chemicals in aqueous environment. Stable aggregates have a fullerene-rich core and a chemical-rich surface layer, and while essentially all C₆₀ molecules aggregate together, a portion of organic molecules remains in water.     

The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides

Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.     

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.