A recombinant malaria protein that can induce Th1 and CD8+ T cell responses without antibody formation.

Inbred strains of mice were immunized with p190-3, a 38-kDa recombinant protein derived from p190, a major merozoite surface Ag of the malaria parasite Plasmodium falciparum. Ag-specific proliferative T cell responses were obtained in H-2b, H-2d, and H-2k mouse strains. Surprisingly, mice of the H-2b haplotype (e.g., C57BL/6) did not give a measurable antibody response to the recombinant protein administered in Freund's adjuvant, but CD8+/CD4- as well as CD4+/CD8- T cells specific for p190-3 could be obtained after in vivo priming and in vitro selection with Ag. Distinct epitopes of p190-3 recognized by the CD8+ and CD4+ T cells from C57BL/6 mice were identified. The CD8+ T cells could kill H-2b APC in the presence of the appropriate epitope-containing peptide. The p190-3-specific CD4+ cells isolated from C57BL/6 mice were of the Th1 type. In contrast, Th2 cells, but no CD8+ T cells were present in a p190-3-specific line from BALB/c mice, which give good antibody responses to p190-3.     

Theory of electrostatic effects in soft biological interfaces using atomic force microscopy.

We calculated the electrostatic force between a planar interface, such as a planar-supported lipid bilayer membrane, and the tip of a stylus on which another lipid bilayer or some other biomacromolecular system might be deposited. We considered styli with rounded tips as well as conical tips. To take into account the effect of dynamical hydrogen-bonded structures in the aqueous phase, we used a theory of nonlocal electrostatics. We used the Derjaguin approximation and identified the systems for which its use is valid. We pointed out where our approach differs from previous calculations and to what extent the latter are inadequate. We found that 1) the nonlocal interactions have significant effects over distances of 10-15 A from the polar zone and that, at the surface of this zone, the effect on the calculated force can be some orders of magnitude; 2) the lipid dipoles and charges are located a distance L from the hydrophobic layer in the aqueous medium and this can have consequences that may not be appreciated if it is ignored; 3) dipoles, located in the aqueous region, can give rise to forces even though the polar layer is unchanged, and if this is ignored the interpretation of force data can be erroneous if an attempt is made to rationalize an observed force with a knowledge of an uncharged surface; 4) the shape of the stylus tip can be very important, and a failure to take this into account can result in incorrect conclusions, a point made by other workers; and 5) when L is nonzero, the presence of charges and dipoles can yield a force that can be nonmonotonic as a function of ionic concentration.     

Atomic force microscope measurements of long-range forces near lipid-coated surfaces in electrolytes.

The interaction of DMPC (L-alpha-dimyristoyl-1,2-diterradecanoyl-sn-glycero-3-phosphoch oli ne, C36H72NO8P) lipid-coated Si3N4 surfaces immersed in an electrolyte was investigated with an atomic force microscope. A long-range interaction was observed, even when the Si3N4 surfaces were covered with nominally neutral lipid layers. The interaction was attributed to Coulomb interactions of charges located at the lipid surface. The experimental force curves were compared with solutions for the linearized as well as with exact solutions of the Poisson-Boltzmann equation. The comparison suggested that in 0.5 mM KCl electrolyte the DMPC lipids carried about one unit of charge per 100 lipid molecules. The presence of this surface charge made it impossible to observe an effective charge density recently predicted for dipole layers near a dielectric when immersed in an electrolyte. A discrepancy between the theoretical results and the data at short separations was interpreted in terms of a decrease in the surface charge with separation distance.     

Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation

Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation, a process known as the bystander effect. Bystander effects can also lead to chromosomal/genomic instability within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The factors that mediate this cellular communication can be transferred between cells via gap junctions or released into the extracellular media following irradiation, but their nature has not been fully characterized. In this study we tested the hypothesis that the bystander effect mediator contains an RNA molecule that may be carried by exosomes. MCF7 cells were irradiated with 2 Gy of X rays and the extracellular media was harvested. RNase treatment abrogated the ability of the media to induce early and late chromosomal damage in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the bystander effect, and genomic instability, are at least in part mediated by exosomes and implicate a role for RNA.     

The lateral distribution of cholesterol in the plane of lipid multibilayers

We consider three models of cholesterol distribution in the plane of a bilayer of DMPC. We analyse recent 2H-NMR data obtained from deuterated fluorescent probes and show that, on the characteristic time-scale of 2H-NMR, it is in accord with a random distribution of cholesterol in a fluid-like DMPC bilayer in a single phase at least for T greater than or approximately equal to 35 degrees C and for 0 less than or equal to c less than or equal to 0.42.     

Effect of integral proteins upon bilayer permeability to small ions

We have modelled the effect of integral proteins upon the permeability of a lipid bilayer membrane to small ions. Our intention is to predict the temperature-and protein concentration-dependence of the relative ionic diffusion coefficient if it is assumed that the ions diffuse entirely through the lipid regions of the membrane, and do not cross the bilayer either by moving through the protein or along its hydrophobic surface. We used models of lipid-protein bilayers that have been used successfully before, together with a modification of the (protein-free) permeability mechanism of Kanchisa, Tsong, Cruzeiro-Hansson and Mouritsen (KTCM). We used Monte Carlo techniques to simulate the thermodynamics and permeability of the membrane because such a calculation, which depends upon spatial and time correlations, cannot be done using a mean field approximation. We took the protein to be analogous to bacteriorhodopsin and calculated the relative diffusion coefficients, R, of small ions through a DMPC bilayer containing such proteins. We found that although there is only a small change in R when T > T _m , R can change by an order of magnitude, depending upon the protein concentration, for T < T _m . We showed that regions of largest lipid area fluctuations and, hence, compressibility occur (i) at the interfaces between regions (which may be very small and not macroscopic phases) of extended-chain and excited-chain lipids, thus showing the similarity between this model and the KTCM model, and (ii) in regions where the temperature is close to T _m but the lipids are prevented from becoming ordered because of the proximity of proteins which try to keep them in their excited-chain states. We have plotted lipid fluctuation and ionic permeability maps to show which regions of the membrane display the largest permeability, and we show how these regions change as a function of temperature. Offprint requests to D. A. Pink