An aryleneethynylene fluorophore for cell membrane staining

The use of an amphiphilic aryleneethynylene fluorophore as a plasma membrane marker in fixed and living mammalian cells and liposome model systems is demonstrated. We show here that the optical properties of the novel dye are almost independent on pH, in the range 5.0-8.0. Spectroscopic characterization performed on unilamellar liposomes ascertained that the fluorescence intensity of the aryleneethynylene fluorophore greatly increases after incorporation in lipidic membranes. Experiments performed on different mammalian cells demonstrated that the novel membrane marker exhibits fast staining and a good photostability that make it a suitable tool for live cell imaging. Importantly, the aryleneethynylene fluorophore was also shown to be a fast and reliable blue membrane marker in classical multicolor immunofluorescence experiments. This study adds new important findings to the recent exploitation of the wide class of aryleneethynylene molecules as luminescent markers for biological investigations.     

Electron-dense material in the cell sall/plasma membrane interface of specialized cortical cells of peanut nodules.

The nitrogen-fixing peanut root nodules have 2-3 layers of specialized cortical cells surrounding the infected zone. These cells are morphologically distinct from the other cortical cells due to the presence of prominent amyloplasts, lipid bodies (oleosomes), large microbodies and localized pockets of electron-dense material at the plasma membrane-cell wall interface. The material is resistant to solubilization by hexane and chloroform. Electron density is enhanced by p-phenylenediamine staining and heavy metal ions such as iron, uranium and osmium. The silver proteinate staining method for suberin has shown positive results. Peroxidative activity can be demonstrated by diaminobenzidine reaction. The material appears to be proteinaceous, but could be complexed with suberin or phenolics. Although the exact nature of this material remains to be clarified, its unique position in the cells of specialized tissue of the cortex relates it to the regulation of bidirectional transport of gases and solutes during symbiosis in the root nodules.     

An ecto-ATPase of thyroidal cell membrane

The isolated cells were obtained from hog thyroid glands treated with dispase. More than 95% of the cells obtained were intact and viable immediately after preparation, and the cell viability did not change during incubation in the experimental conditions. ATP added to the external medium of whole cell suspensions was hydrolyzed in the presence of various divalent cations, especially Mg, and the rate of hydrolysis of ATP was not significantly different between the Mg-ion system and the completed ion system (Mg+Na+K). When whole cell suspensions were disrupted with homogenizer, the hydrolysis of ATP was markedly increased by adding Na plus K. But there was no difference in the Mg-ion system between cell homogenates and whole cell suspensions. ADP, AMP and adenosine as reaction products were found in the reaction mixture which resulted from the hydrolysis of ATP by whole cell suspensions. Our data suggest that Mg-ATPase in the thyroidal isolated cells is an ectoenzyme whose active site(s) are exposed to the external surface of plasma membrane, and that ATP is finally hydrolyzed to adenosine via ADP and AMP by the enzyme(s).     

Stress-free state of the red blood cell membrane and the deformation of its skeleton

The response of a red blood cell (RBC) to deformation depends on its membrane, a composite of a lipid bilayer and a skeleton, which is a closed, twodimensional network of spectrin tetramers as its bonds. The deformation of the skeleton and its lateral redistribution are studied in terms of the RBC resting state for a fixed geometry of the RBC, partially aspirated into a micropipette. The geometry of the RBC skeleton in its initial state is taken to be either two concentric circles, a references biconcave shape or a sphere. It is assumed that in its initial state the skeleton is distributed laterally in a homogeneous manner with its bonds either unstressed, presenting its stress-free state, or prestressed. The lateral distribution was calculated using a variational calculation. It was assumed that the spectrin tetramer bonds exhibit a linear elasticity. The results showed a significant effect of the initial skeleton geometry on its lateral distribution in the deformed state. The proposed model is used to analyze the measurements of skeleton extension ratios by the method of applying two modes of RBC micropipette aspiration.     

Mechanisms of membrane deformation

Membrane traffic requires the generation of high-curvature lipid-bound transport carriers represented by tubules and vesicles. The mechanisms through which membranes are deformed has gained much recent attention. A major advance has been the demonstration that direct interactions between cytosolic proteins and lipid bilayers are important in the acquisition of membrane curvature. Rather than being driven only by the formation of membrane-associated structural scaffolds, membrane deformation requires physical perturbation of the lipid bilayer. A variety of proteins have been identified that directly bind and deform membranes. An emerging theme in this process is the importance of amphipathic peptides that partially penetrate the lipid bilayer.     

Bioelectrorheological model of the cell. 3. Viscoelastic shear deformation of the membrane.

An analytical electromechanical model of a spherical cell exposed to an alternating electric field was used to calculate shear stress generated in the cellular membrane. Shape deformation of Neurospora crassa (slime) spheroplasts was measured. Statistical analysis permitted empirical evaluation of creep of the cellular membrane within the range of infinitesimal stress. Final results were discussed in terms of various rheological models.     

Studies of a large transformation-increased membrane protein in the BHK21 cell system

We have recently described in BHK cells a plasma membrane protein of molecular weight 177,000, which is significantly increased in Hamster Sarcoma Virus-transformed cells (Lage-Davila, A. and Montagnier, L. (1977) Biochem. Biophys. Res. Commun. 79, 577--584). We present now a study of proteins from purified plasma membrane fractions in the same pair of clones. Solubilization conditions, cross-linking experiments, metabolic labelling and enzymatic radioiodination allow to characterize this 177,000 transformation-increased protein as an integral membrane glycoprotein partially exposed at the outer cell surface. Additional information on other membrane proteins in this system is also given.     

Studies of a large transformation-increased membrane protein in the BHK21 cell system

We have recently described in BHK cells a plasma membrane protein of molecular weight 177 000, which is significantly increased in Hamster Sarcoma Virus-transformed cells (Lage-Davila, A. and Montagnier, L. (1977) Biochem. Biophys. Res. Commun. 79, 577–584). We present now a study of proteins from purified plasma membrane fractions in the same pair of clones. Solubilization conditions, cross-linking experiments, metabolic labelling and enzymatic radioiodination allow to characterize this 177 000 transformation-increased protein as an integral membrane glycoprotein partially exposed at the outer cell surface. Additional information on other membrane proteins in this system is also given.     

An oscillatory mechanism for the gamma-glutamyl transpeptidase-mediated translocation of amino acids across the cell membrane

The reinterpretation of the kinetics of the gamma-glutamyl cycle-mediated uptake of amino acids in the light of the cycle's wave mechanical properties shows that its oscillatory periods are modulated by the chemical nature and the concentrations of amino acids. The periods of the cycle are the half-lives of glutathione whose function is to synchronize the oscillations of the two pathways of the cycle. gamma-glutamyl transpeptidase, an amphipathic membrane protein and the master oscillator of the cycle degrades glutathione and translocates amino acids in a discontinuous manner suggesting that it flip-flops across the membrane, the periods of the flip-flops being the oscillatory periods of the gamma-glutamyl transpeptidase/amino acid complexes. The energies of the flip-flops are quantized and independent of metabolic energy. The principal quantum numbers are dependent on the amino acids being translocated. In their translocation, those amino acids which are good substrates of the gamma-glutamyl transpeptidase possess higher principal quantum numbers than those which are poor substrates, an observation which gives support to the flip-flopping of the gamma-glutamyl transpeptidase/amino acid complexes across the cell membrane.     

An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes

Fas, a member of the tumor necrosis factor receptor family, can upon ligation by its ligand or agonistic antibodies trigger signaling cascades leading to cell death in lymphocytes and other cell types. Such signaling cascades are initiated through the formation of a membrane death-inducing signaling complex (DISC) that includes Fas, the Fas-associated death domain protein (FADD) and caspase-8. We report here that a considerable fraction of Fas is constitutively partitioned into sphingolipid- and cholesterol-rich membrane rafts in mouse thymocytes as well as the L12.10-Fas T cells, and Fas ligation promotes a rapid and specific recruitment of FADD and caspase-8 to the rafts. Raft disruption by cholesterol depletion abolishes Fas-triggered recruitment of FADD and caspase-8 to the membrane, DISC formation and cell death. Taken together, our results provide the first demonstration for an essential role of membrane rafts in the initiation of Fas-mediated cell death signaling.     

An Eulerian path approach to global multiple alignment for DNA sequences.

With the rapid increase in the dataset of genome sequences, the multiple sequence alignment problem is increasingly important and frequently involves the alignment of a large number of sequences. Many heuristic algorithms have been proposed to improve the speed of computation and the quality of alignment. We introduce a novel approach that is fundamentally different from all currently available methods. Our motivation comes from the Eulerian method for fragment assembly in DNA sequencing that transforms all DNA fragments into a de Bruijn graph and then reduces sequence assembly to a Eulerian path problem. The paper focuses on global multiple alignment of DNA sequences, where entire sequences are aligned into one configuration. Our main result is an algorithm with almost linear computational speed with respect to the total size (number of letters) of sequences to be aligned. Five hundred simulated sequences (averaging 500 bases per sequence and as low as 70% pairwise identity) have been aligned within three minutes on a personal computer, and the quality of alignment is satisfactory. As a result, accurate and simultaneous alignment of thousands of long sequences within a reasonable amount of time becomes possible. Data from an Arabidopsis sequencing project is used to demonstrate the performance.     

Nonlinear large deformation of a spherical red blood cell induced by ultrasonic standing wave

Biomechanics and Modeling in Mechanobiology - A computational model is developed to investigate the nonlinear static deformation of a spherical (osmotically swollen) red blood cell (RBC) induced by...     

An ASIC-chip for stereoscopic depth analysis in video-real-time based on visual cortical cell behavior

In a stereoscopic system both eyes or cameras have a slightly different view. As a consequence small variations between the projected images exist ("disparities") which are spatially evaluated in order to retrieve depth information. We will show that two related algorithmic versions can be designed which recover disparity. Both approaches are based on the comparison of filter outputs from filtering the left and the right image. The difference of the phase components between left and right filter responses encodes the disparity. One approach uses regular Gabor filters and computes the spatial phase differences in a conventional way as described already in 1988 by Sanger. Novel to this approach, however, is that we formulate it in a way which is fully compatible with neural operations in the visual cortex. The second approach uses the apparently paradoxical similarity between the analysis of visual disparities and the determination of the azimuth of a sound source. Animals determine the direction of the sound from the temporal delay between the left and right ear signals. Similarly, in our second approach we transpose the spatially defined problem of disparity analysis into the temporal domain and utilize two resonators implemented in the form of causal (electronic) filters to determine the disparity as local temporal phase differences between the left and right filter responses. This approach permits video real-time analysis of stereo image sequences (see movies at http://www.neurop.ruhr-uni-bochum.de/Real- Time-Stereo) and a FPGA-based PC-board has been developed which performs stereo-analysis at full PAL resolution in video real-time. An ASIC chip will be available in March 2000.     

An ATP pool with rapid turnover within the cell membrane

Seven-day-old cultures of rat leg muscle cells were double labelled by addition of [¹⁴C] adenine in the culture medium (2 hrs 15 mins) and followed by addition of [³²P] phosphate (15 min). The specific activity (S.A.) of the isolated cyclic [¹⁴C] adenine 3′ – 5′ monophosphate (cAMP) was similar to that of the bulk ATP. The S.A. of [³²P] from cAMP was, however, higher than that of bulk ATP. The S.A. of [³²P] from cAMP could be further modified by prevention of normal muscle cell fusion. It is probable that the cAMP with high [³²P] S.A. was synthesized from a cell membrane pool of ATP with rapid turnover.     

Analyzing the interplay between single cell rheology and force generation through large deformation finite element models

In this study, experimental results of single cell spreading between two parallel microplates are exploited through finite element modeling. Axisymmetric computations at finite strains are performed to extract the mechanical properties of the cell which can account for cell shape evolution and traction force generation. Our model includes two distinct components representing the cortex associated with the bilayer membrane on the one hand, and the rest of the cell on the other hand. The former is modeled as a homogeneous hyperelastic material described by a slightly compressible Gent strain energy function, while the latter is idealized either as a quasi-incompressible Newtonian fluid or as another homogeneous hyperelastic material. The kinetics of spreading is ensured by a stapling procedure defined from experimental observations. Material parameters are then optimized to match the simulation closely with the experimental data. In particular, the elastic modulus of the cortex is estimated at about 1,000?Pa in both models, while the cell interior is characterized by a viscosity of 1,000?Pa.s in the biphasic model, or by an elastic modulus of about 100?Pa in the hyperelastic one. These results are in good agreement with G(') and G(') measurements carried out previously in the same parallel plates setup and estimated at the typical rate of cell straining. Moreover, stresses are found to concentrate at the edge of the cell-substrate contact area. These observations allow explaining the relationship between cell spreading and force increase since spreading and the consequent straining of the cell mechanical structure could be the source of the force applied by the cell on its substrate. They could also explain, in a very simple manner, why force-sensitive focal contacts concentrate at the cell edges.     

An Eulerian model of phytoplankton photosynthetic response in the upper mixed layer

An Eulenan model of an upper mixed layer forced by dynamic andconvective processes is combined with a temporally responsivephotoadaptation model of photosynthesis. Incident radiationis divided into photosyntheticalJy active and inactive componentsto distinguish between biological and physical forcing. Thephysical model is initialized to simulate conditions in LakeTiticaca on August 12, 1982 Model output provides the time courseof water column temperature, phytoplankton photoinhibition andrelative phytoplankton carbon fixation as depth contours forcomparison with published observations. The predicted and observedtemperature profiles generally agree; diurnal changes in specifichumidity are required to more accurately specify heat loss.The predicted photoinhibition resembles the observed photosyntheticstate of the natural community based on fluorescence, but themodeled recovery from photoinhibition is too rapid. This discrepancyalso affects the comparison between predicted and observed bottledeterminations of carbon fixation. The basic formulations inthe model provide approximate fits to field observations. Ifmore complete information on physical and biological initializationsand on temporal forcing were available, the basic formulationscould be refined