Vitamin E Induces Phosphatidylserine Externalization and Red Cell Adhesion to Endothelial Cells

Red blood cell (RBC) adhesion to vessel wall endothelium is a potent catalyst of vascular occlusion and occurs in oxidative stress states such as hemoglobinopathies and cardiovascular conditions. These are often treated with vitamin E (VitE), a “classic” antioxidant. In this study, we examined the effects of VitE on RBC adhesion to vascular endothelial cells (EC), and on translocation of phosphatidylserine (PS) to RBC surface, known as a potent mediator of RBC/EC adhesion, facilitating thrombus formation. Treatment of RBC with VitE strongly induces (up to sevenfold) PS externalization and enhances (up to 20-fold) their adherence to EC. The VitE hydrophilic analogue—Trolox—does not incorporate into cell membranes. Trolox did not exhibit any of these effects, implying that the VitE effect is due to its known ability to incorporate into cell membranes. The membrane-incorporated VitE significantly reduced the level of reactive oxygen species in H₂O₂-treated RBC, demonstrating that VitE elevates RBC/EC adhesion despite acting as an anti-oxidant. This study demonstrates for the first time that contrary to the common view of VitE as a beneficial supplement, VitE may introduce a circulatory risk by inducing flow-disturbing RBC adherence to blood vessel wall and the pro-thrombotic PS exposure.     

The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.     

Staff Nurses’ Perceptions and Experiences about Structural Empowerment: A Qualitative Phenomenological Study

The aim of the study reported in this article was to investigate staff nurses’ perceptions and experiences about structural empowerment and perceptions regarding the extent to which structural empowerment supports safe quality patient care. To address the complex needs of patients, staff nurse involvement in clinical and organizational decision-making processes within interdisciplinary care settings is crucial. A qualitative study was conducted using individual semi-structured interviews of 11 staff nurses assigned to medical or surgical units in a 600-bed university hospital in Belgium. During the study period, the hospital was going through an organizational transformation process to move from a classic hierarchical and departmental organizational structure to one that was flat and interdisciplinary. Staff nurses reported experiencing structural empowerment and they were willing to be involved in decision-making processes primarily about patient care within the context of their practice unit. However, participants were not always fully aware of the challenges and the effect of empowerment on their daily practice, the quality of care and patient safety. Ongoing hospital change initiatives supported staff nurses’ involvement in decision-making processes for certain matters but for some decisions, a classic hierarchical and departmental process still remained. Nurses perceived relatively high work demands and at times viewed empowerment as presenting additional. Staff nurses recognized the opportunities structural empowerment provided within their daily practice. Nurse managers and unit climate were seen as crucial for success while lack of time and perceived work demands were viewed as barriers to empowerment.     

Localization by in situ hybridization of a low copy chimaeric resistance gene introduced into plants by direct gene transfer

Summary An in situ hybridization method was developed for detecting single or low copy number genes in metaphase chromosomes of plants. Using as a probe ³H-labelled plasmid pABDI, which confers kanamycin resistance (Km^r) to transformed cells. DNA introduced into the plant genome by direct gene transfer was detected with a high efficiency: about 60% to 80% of interphase and metaphase plates showed a strong signal. The insertion site of the Km^r gene in two independent transformants was localised on different homologous chromosome pairs. This result independently confirmed previous genetic data which had indicated that transformed DNA was integrated into plant chromosomes in single blocks.     

Electrical fusion of protoplasts from sycamore mutants and hybrid selection on hypoxanthine-aminopterin-thymidine (HAT) medium

Summary An electrical fusion method has been used to form somatic hybrids between protoplasts of two mutant cell lines of sycamore tissue culture cells. Both mutants will not grow in a hypoxanthine-aminopterin-thymidine (HAT) medium. It was possible to select the fused hybrids from homospecific fusion products and nonfused protoplasts by the use of HAT medium. In this way the viability and regeneration of the fused cells during the first few weeks of culture could be evaluated. An electron microscopic examination of the fusion process showed that it occurred at a series of points along the surface of the plasmalemma. Cytoplasmic bridges between the two cells were formed separated by vesicles which later dispersed to give complete cytoplasmic continuity between the cells.     

The actions of Ca2+ ionophores on rat basophilic (2H3) cells are dependent on cellular ATP and hydrolysis of inositol phospholipids. A comparison with antigen stimulation

Calcium-specific ionophores are used widely to stimulate Ca2+-dependent secretion from cells on the assumption that permeabilization of the cell membranes to Ca2+ ions leads to a rise in concentration of cytosolic Ca2+ ([Ca2+]i), which in turn serves as a signal for secretion. In this way, events that precede mobilization of Ca2+ ions via receptor stimulation are bypassed. One such event is thought to be the rapid hydrolysis of membrane inositol phospholipids to form inositol phosphates and diacylglycerol. Accordingly, rat leukemic basophil (2H3) cells can be stimulated to secrete histamine either with the ionophores or by aggregation of receptors for IgE in the plasma membrane. We find, however, that ionophore A23187 stimulates secretion of histamine only at concentrations (200-1000 nM) that stimulate hydrolysis of membrane inositol phospholipids. The extent of hydrolysis of inositol phospholipids was dependent on the concentration of ionophore and the presence of external Ca2+ ions and correlated with the magnitude of the secretory response. A similar correlation between secretion and hydrolysis of inositol phospholipids was observed in response to the Ca2+-specific ionophore, ionomycin. Although this hydrolysis (possibly a consequence of elevated [Ca2+]i) was less extensive than that induced by aggregation of receptors, it may govern the secretory response to A23187. The studies revealed one paradox. The rise in [Ca2+]i depended on intracellular ATP levels, when either an ionophore or antigen was used as a stimulant irrespective of whether hydrolysis of inositol phospholipids was stimulated or not. The concept of how the ionophores act, therefore, requires critical reevaluation.     

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.