Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.     

Lysolipids do not inhibit influenza virus fusion by interaction with hemagglutinin

The interaction of a spin-labeled lysophosphatidylcholine analog with intact and bromelain-treated influenza viruses as well as with the bromelain-solubilized hemagglutinin ectodomain has been studied. The inhibition of fusion of influenza viruses with erythrocytes by the lysophosphatidylcholine analog was similar to that observed for non-labeled lysophosphatidylcholine. Only a weak interaction of the lysophosphatidylcholine analog with the hemagglutinin ectodomain was observed even upon triggering the conformational change of the ectodomain at a low pH. A significant interaction of spin-labeled lysophosphatidylcholine with the hemagglutinin ectodomain of intact viruses was observed neither at neutral nor at low pH, whereas a strong interaction of the lipid analog with the viral lipid bilayer was evident. We suggest that the high number of lipid binding sites of the virus bilayer and their affinity compete efficiently with binding sites of the hemagglutinin ectodomain. We conclude that the inhibition of influenza virus fusion by lysolipids is not mediated by binding to the hemagglutinin ectodomain, preventing its interaction with the target membrane. The results unambiguously argue for an inhibition mechanism based on the action of lysolipid inserted into the lipid bilayer.     

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

Abstract: The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TER) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one-to-one mixtures of these proteins. Moreover, a one-to-one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (∼350 Ω× cm²) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one-to-one combinations of these proteins elevated transcellular resistances significantly (2.3- to 2.9-fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (∼1,000 Ω× cm²) could, by contrast, be increased only 1.1- to 1.2-fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood-brain barrier.     

pH-dependent binding of the fluorophore bis-ANS to influenza virus reflects the conformational change of hemagglutinin

Binding of the fluorophore 1,1-bis(4-anili-no) naphthalene-5,5-disulfonic acid (bis-ANS) to influenza virus A/PR 8/34 is strongly enhanced at low pH. Binding is accompanied by a significant increase in fluorescence intensity. The binding and the fluorescence increase are associated with the low-pH induced conformational change of the viral spike protein, hemagglutinin, exposing hydrophobic binding sites. The data indicate that in addition to the hydrophobic N-terminus of HA2 other hydrophobic sequences of the HA ectodomain become accessible to bis-ANS at low pH. It is shown that the time course of the fluorescence increase of bis-ANS at low pH is determined by the conformational change of HA. The application of this assay for continuously monitoring the kinetics of the structural alteration in HA is discussed and its relevance for elucidating the temporal relationship between the conformational change of HA and virus-membrane fusion is outlined.Abbreviations HA hemagglutinin - BHA bromelain-solubilized ectodomain of HA - N-HA2 N-terminus of the HA2 subunit - PBS phosphate buffered saline - bis-ANS (1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid) - R18 octadecylrhodamine B chloride - FDQ fluorescence dequenching - RBC red blood cell Correspondence to: A. Herrmann     

Headgroup-specific exposure of phospholipids in ABCA1-expressing cells

ABCA1 has been established to be required for the efflux of cholesterol and phospholipids to apolipoproteins such as apoA-I. At present, it is unclear whether ABCA1-mediated lipid exposure is specific with regard to lipid headgroups and whether it requires calcium activation and the presence of a lipid acceptor. In the present work, we found exofacial exposure of endogenous phosphatidylserine in the absence of apoA-I to be enhanced in ABCA1-GFP expressing MDCKII and HeLa cells compared with control cells. By using C6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phospholipid analogues, we observed elevated redistribution of phosphatidylserine and phosphatidylethanolamine but not of phosphatidylcholine analogues from the cytoplasmic to the exoplasmic leaflet of the plasma membrane of ABCA1-GFP expressing cells. Whereas glyburide affected neither the level of exofacial endogenous PS nor the outward movement of the amino phospholipid analogues, the latter was sensitive to intracellular Ca2+ in ABCA1-GFP expressing cells, further enhancing outward analogue redistribution with respect to control cells. Both receptor-mediated endocytosis and fluidphase endocytosis were reduced in MDCKII cells expressing ABCA1-GFP. Glyburide raised the level of receptor-mediated endocytosis in the ABCA1-GFP expressing cell to the level of control cells in the absence of glyburide. In control cells, however, fluid-phase endocytosis but not receptor-mediated endocytosis was significantly reduced upon glyburide treatment.     

Development of the HKHbios: a new biotic score to assess the river quality in the Hindu Kush-Himalaya

Within the ASSESS-HKH project (Development of an Assessment System to Evaluate the Ecological Status of Rivers in the Hindu Kush-Himalayan (HKH) region—a research project funded by the European Union; contract number: INCO-CT-2005-003659) a benthic invertebrate-based scoring system (HKHbios; Hindu Kush-Himalayan biotic score) was developed. The development was based on multi-habitat samples from 198 sampling sites located in five ecoregions and five Asian countries (Bangladesh, Bhutan, India, Nepal and Pakistan) taken in two different seasons (pre- and post-monsoon). Environmental and biological screening data were used to select macro-invertebrates as indicators for the ecological river quality. Taxa scores were assigned based on the range and distribution patterns of taxa amongst different degrees of impact and on available autecological information. In total, 199 taxa were scored for the HKHbios, which is calculated a weighted average score per taxon (ASPT). The range of the index values under different degrees of stress was evaluated and a five-class quality assessment system was generated for each ecoregion. Correlation analysis between the HKHbios, 38 selected environmental parameters and complex PCA gradients were used to test the response of the HKHbios to different kinds of impact.     

Determination of physostigmine in plasma by high-performance liquid chromatography and fluorescence detection

Physostigmine (PHY) is an anticholinergic drug used in the treatment of neuromuscular disorders and organophosphate poisoning. We described a sensitive, accurate, and reproducible method for PHY determination in biological materials. The method utilized a liquid/liquid, ion pair extraction, normal phase HPLC separation, and fluorometric quantitation at 240 nm excitation and 360 nm emission wavelength. We used neostigmine as a stabilizing agent to protect PHY from degradation and dimethylphysostigmine as an internal standard. The peak-height ratio vs concentration was linear over a working range from 0.50 to 25.0 ng/ml of PHY in plasma. Sensitivity of the method was 100 pg/ml of plasma which was the limit of quantitative detection under the experimental conditions used. Precision of the method was evaluated using plasma spiked with two concentrations of PHY: 1.0 and 10.0 ng/ml. Intra-day coefficient of variation (CV) ranged from 3.8 to 5.3%, and inter-day CV ranged from 1.8 to 3.6% for the two levels. The average recovery was 92%. We applied the method to examine the stability of PHY in plasma stored at -15 and -80 degrees C. The data indicated that PHY can be stored at either temperature for 9 weeks without undergoing significant alterations.     

Genetic mapping of the early responses to salt stress in Arabidopsis thaliana

Salt stress decreases plant growth prior to significant ion accumulation in the shoot. However, the processes underlying this rapid reduction in growth are still unknown. To understand the changes in salt stress responses through time and at multiple physiological levels, examining different plant processes within a single set-up is required. Recent advances in phenotyping has allowed the image-based estimation of plant growth, morphology, colour and photosynthetic activity. In this study, we examined the salt stress-induced responses of 191 Arabidopsis accessions from 1 h to 7 days after treatment using high-throughput phenotyping. Multivariate analyses and machine learning algorithms identified that quantum yield measured in the light-adapted state (F_v′/F_m′) greatly affected growth maintenance in the early phase of salt stress, whereas the maximum quantum yield (QY_max) was crucial at a later stage. In addition, our genome-wide association study (GWAS) identified 770 loci that were specific to salt stress, in which two loci associated with QY_max and F_v′/F_m′ were selected for validation using T-DNA insertion lines. We characterized an unknown protein kinase found in the QY_max locus that reduced photosynthetic efficiency and growth maintenance under salt stress. Understanding the molecular context of the candidate genes identified will provide valuable insights into the early plant responses to salt stress. Furthermore, our work incorporates high-throughput phenotyping, multivariate analyses and GWAS, uncovering details of temporal stress responses and identifying associations across different traits and time points, which are likely to constitute the genetic components of salinity tolerance.