Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules

Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.     

Red cell membrane elasticity as determined by flow channel technique.

The elasticity of red cell membrane was determined in a rectangular flow channel under controlled shear flow. The relation between shear stress and cell extension ratio (lambda) has been analyzed with the use of Evans' two-dimensional model. The deformed cell shapes observed experimentally agreed well with the model with lambda up to 1.4. The best correlation was found at lambda = 1.2. The analysis suggests a nonlinear extensional membrane modulus in the low stress range encountered in the flow channel. In terms of an appropriate strain parameter, the elastic modulus is shown to rise toward the level encountered in micropipette aspiration experiments. The implications of the present findings in modeling of cell mechanics and in cell hemolysis are discussed.     

Induction of CD4+ cytotoxic T cells by sensitization with allogeneic melanomas bearing shared or cross-reactive HLA-A.

Human melanoma is an immunogenic neoplasm whereby enhancement of specific cell-mediated immunity can alter tumor progression. HLA-A2-restricted CTL have been demonstrated to kill allogeneic HLA-A2-matched melanoma. We investigated the ability of allogeneic melanoma cells sharing HLA-A antigens to sensitize melanoma patients' lymphocytes to induce HLA-A-restricted CTL to autologous melanoma. PBL from melanoma patients were cocultured with autologous melanoma cells in defined "cocktail medium" to generate melanoma-specific HLA-A-restricted CTL lines. CTL generated by sensitization with allogeneic melanoma bearing shared HLA-A2, A11, A24, or "cross-reactive" HLA-A antigens could kill almost as many autologous melanoma cells as CTL sensitized with autologous melanoma. There are HLA-A antigens that are immunogenically cross-reactive because they share determinant epitopes. CTL were not activated NK or LAK cells. The HLA restriction and melanoma cell specificity of the CTL were demonstrated by cold target inhibition with autologous and allogeneic melanoma and B lymphoblasts. Anti-CD3 and anti-HLA AB inhibited CTL killing of melanoma. The CTL were predominantly CD3+CD4+ TCR alpha/beta+. These studies demonstrate that melanomas being shared or cross-reactive HLA-A can be used for in vitro generation of HLA-restricted CTL that recognize melanoma-associated antigens. The findings have very important implications in human tumor immunotherapy.     

Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.     

Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes (8 x 10(6) molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis (7 x 10(7) molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range (2.5 x 10(8) to 3.5 x 10(8) molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10(8) interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.