Peter G. Petrov

A Mechanism of Release of Calreticulin from Cells During Apoptosis

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca(2+) homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca(2)(+)-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.

Effect of Hydroperoxides on Red Blood Cell Membrane Mechanical Properties

We investigate the effect of oxidative stress on red blood cell membrane mechanical properties in vitro using detailed analysis of the membrane thermal fluctuation spectrum. Two different oxidants, the cytosol-soluble hydrogen peroxide and the membrane-soluble cumene hydroperoxide, are used, and their effects on the membrane bending elastic modulus, surface tension, strength of confinement due to the membrane skeleton, and 2D shear elastic modulus are measured. We find that both oxidants alter significantly the membrane elastic properties, but their effects differ qualitatively and quantitatively. While hydrogen peroxide mainly affects the elasticity of the membrane protein skeleton (increasing the membrane shear modulus), cumene hydroperoxide has an impact on both membrane skeleton and lipid bilayer mechanical properties, as can be seen from the increased values of the shear and bending elastic moduli. The biologically important implication of these results is that the effects of oxidative stress on the biophysical properties, and hence the physiological functions, of the cell membrane depend on the nature of the oxidative agent. Thermal fluctuation spectroscopy provides a means of characterizing these different effects, potentially in a clinical milieu.

Raman and X-ray photoelectron spectroscopy study of carbon nitride thin films

Abstract Carbon nitride thin films were deposited on Si(100) substrates by electron beam evaporation of graphite and simultaneous low energy nitrogen ion bombardment. They were analysed by Raman and X-ray photoelectron spectroscopy. The formed amorphous layers are tetrahedrally bonded and consist of sp3 carbon bonds with one nitrogen atom among its nearest neighbours. Substitution of the tetrahedrally bonded carbon atom by nitrogen leads to decrease of the percentage weight of the nanocrystalline diamond phase and formation of a CNx phase embedded in the amorphous carbon layer. By changing the deposition conditions, redistribution of sp2 and sp3 bonded C–N occurs.

The effect of oxidative stress on the membrane dipole potential of human red blood cells

The membrane dipole potential (ψ(d)) is an important biophysical determinant of membrane function and a sensitive indicator of lipid organisation. In this study we have used the environmentally sensitive probe di-8-anepps to explore the effects of oxidative stress on the membrane dipole potential of human erythrocytes. Cells suspended in 0.15mM phosphate buffered saline containing 0.1mg/ml albumin maintained a mean value for ψ(d) of 270 (±20) mV over the course of 1hour. In the presence of 0.4mM cumene hydroperoxide there was an increase in ψ(d) of 14 (±7)%, accompanied by a decrease in cell diameter of ~14 (±2)%. Exposure of the cells to 0.4mM hydrogen peroxide caused ψ(d) to decrease by 13 (±8)% at the centre of the cell and 8 (±5)% at the edge whilst the diameter remained constant. In both cases the changes were equivalent to a change in transmembrane electric field of a magnitude of ~10MVm(-1), sufficient to influence membrane function. Raman microspectrometry supported the conclusion that cumene exerts its effect primarily on membrane lipids whilst hydrogen peroxide causes the formation of spectrin-haemoglobin complexes which stiffen the membrane.

Forced advancement and retraction of polar liquids on a low energy surface

Abstract Dependences of advancing and receding dynamic contact angles on contact-line velocity during forced spreading and retraction of drops of polar liquids (water and aqueous glycerol solutions) on a low energy polyethylene terephthalate surface are studied. The experimental data for 0a/V and 0r/V are interpreted on the basis of the molecular-kinetic and hydrodynamic models of contact-line motion. It is shown that the molecular-kinetic approach provides the best description of the experimental data in the velocity range studied (1 · 10−4-2 · 10−1 cm s−1) when two sets of molecular parameters are used for the initial and final branches of the kinetic dependences. Blake (T.D. Blake, 1988 AIChE Int. Symp. on the Mechanics of Thin-Film Coating, Paper 1a: Wetting kinetics — how do wetting lines move?, March 6–10, 1088, New Orleans, LA) assumed that such behaviour is due to heterogeneity of the solid surface. The hydrodynamic model predicts physically unreasonable values of its characteristic parameter, the slip length Ls. It is shown that the plateaux in the kinetic dependences 0a/V and 0r/V, established in a previous study represent parts of stretched S-shaped curves with horizontal inflection regions. The significance of the initial slopes preceding the plateaux is confirmed again and it is shown that extrapolation of these initial branches to V = 0 leads to values that coincide well with the static hysteresis angles of advancement and retraction.

Mechanical properties of ternary lipid membranes near a liquid?liquid phase separation boundary

We study the mechanical properties of ternary lipid bilayers assembled in giant vesicles, formed from a saturated and an unsaturated phosphocholine (in equal proportions) and cholesterol. As a function of temperature, these systems can undergo in-plane phase separation. Using image analysis we identify the vesicle contour, and quantify the vesicle shape and the amplitude of membrane thermal fluctuations. The two lipid compositions chosen show different thermotropic behaviours. At 60 mol% cholesterol the membrane is in a uniform liquid state over the entire temperature range investigated (10-50 °C), but vesicles containing 30 mol% cholesterol undergo phase separation into two immiscible liquid phases at around 28 °C. Upon cooling below this transition temperature we observe a marked increase in the measured bending elastic modulus. Phase separation proceeds over a long time (tens of minutes), and we measure the properties of vesicles both during the domain coarsening phase and in the fully phase separated condition. Fluorescence microscopy allows us to identify the coexisting phases. We can therefore measure directly the bending moduli of each of the phases as a function of temperature, showing a strong variation which is attributed to the changing phospholipid and cholesterol composition.

Effect of geometry on steady wetting kinetics and critical velocity of film entrainment

Abstract The velocity dependence of receding dynamic contact, angles θ r / U for siliconized cylinders of different, radii withdrawn from a glycerol-water mixture [19] show an independence on geometry and substantial influence of the material properties of the solid surface. These data are compared with the results of Ngan and Dussan [16a,b] for advancing angles θ α / U (silicon oil displacing air), which suggest a considerable effect of geometry. A similar asymmetry of the effects of geometry and material properties on the critical velocities of liquid and air film entrainment follows from the Juxtaposition of our previous results for u cr R [19,20] with literature data on U cr A onto different solid substrates. The experimental data are interpreted on the basis of the equations of Cox-Voinov [21,22], describing the data of Ngan and Dussan quantitatively. The data for the receding meniscus can be represented quantitatively only by a combined Blake-Haynes-Voinov equation taking into account the dissipation in the three-phase contact zone and in the bulk liquid.

Comparison of the static and dynamic contact angle hysteresis at low velocities of the three-phase contact line

The forced spreading and retraction of a drop with variable volume onto a poly(ethylene terephthalate) surface has been studied. Water and an aqueous glycerol solution were used as liquid phases. The critical static contact angles of advancement θAcr and retraction θRcr at which the contact line motion starts were determined and compared with the values obtained by extrapolation of the velocity dependences of the dynamic advancing, θa, and receding, θr, contact angles to V=0. The dependences of the dynamic advancing and receding contact angles on the contact line velocity were studied in the range 0–3 · 10−3 cm s−1. With the more viscous liquid, θa and θr depended on V up to 1 · 10−3 cm s−1. These parts of the data are well represented by the Blake and Haynes molecular description of the wetting kinetics with equal molecular—kinetic parameters for advancement and retraction. At higher velocities plateaux in both θa(V) and θr(V) kinetic relationships have been observed. The receding dynamic angles of the plateaux differ substantially from the critical static receding angles in both systems studied. In this region the dynamic contact angle hysteresis, although being velocity independent, differs from the static contact angle hysteresis. The value of the dynamic hysteresis tends to the static one at velocities lower than 1 · 10−3 cm s−1, but the two quantities coincide only at V = 0. From the equality of the molecular kinetic parameters at advancement and retraction, it follows that one can determine the Young angle θ0 from a simultaneous measurement of the advancing and receding dynamic contact angles at sufficiently low velocities.

Cell division and cell enlargement in isolated Cucurbita cotyledons grown in darkness and in light

Abstract.The spatial and temporal patterns of post-embryonal cell growth and cell division were characterised in excised cotyledons of vegetable marrow (Cucurbitapepo L. var. giromontia Alef.) incubated in water. The concurrent roles of these two processes in cotyledon growth were determined using paradermal sections of the first palisade layer of developing cotyledons. Tissue specificity was observed in the pattern of cell division. The daughter cells derived from an initial cell, which had already differentiated before imbibition of the seeds, were tightly packed in a cluster, which enabled us to monitor cell division during early cotyledon development. Heterogeneity of cell size was recognised during the process of cell proliferation in the cluster, suggesting that cell division is uncoupled from control of cell size. There was significantly more cell division in the marginal part of the cotyledons than in other parts, suggesting high activity of the marginal meristem. Light enhanced cell and cotyledon enlargement, but had no effect on the number of divisions. This study elucidated the cellular basis of post-germinative Cucurbita cotyledon morphogenesis and development.

Red Blood Cell Susceptibility to Pneumolysin: CORRELATION WITH MEMBRANE BIOCHEMICAL AND PHYSICAL PROPERTIES.

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell.