Circular dichroism of nerve membrane fractions: effects of temperature, pH and electrolytes

Abstract— We have demonstrated significant differences in the circular dichroic (CD) spectra of myelin, synaptic vesicle and synaptosomal membrane preparations from rat brain. Although the measured CD spectra probably contained components attributable to turbidity, we have presented arguments to indicate that turbidity effects could not account for all the observed differences. We think it likely, therefore, that these three membrane fractions exhibit intrinsically different CD spectra, and therefore contain proteins of differing three-dimensional structure. The perturbation of membrane CD produced by electrolytes appeared to involve specific ionic effects. Here again, effects of turbidity were probably involved. The increase in light scattering was demonstrated by an increase in A₂₅₀ at increasing salt concentrations. The increase in A₂₅₀ and the decrease in the intensity of [ø] showed no simple correlation. The effect of acidic pH (below pH 6–5) on the membrane CD appeared similar to that of increasing salt concentrations. In contrast, the effect of basic pH appeared to be one of denaturation. The effect of temperature on the membrane CD was one of stability below or at physiological temperatures and of irreversible instability at temperatures slightly above the physiological range. The CD of synaptosomal membrane preparations changed slowly over prolonged periods at low temperatures or by cycles of freezing and thawing. Our observations indicate that careful attention to the physical and chemical environment of membranes is necessary in CD investigations of membrane preparations.     

Mouse splenic macrophage cell lines with different antigen-presenting activities for CD4+ helper T cell subsets and allogeneic CD8+ T cells.

A panel of seven mouse splenic macrophage cell lines, derived from cloned progenitors, was compared for their ability to present antigen to Th1 or Th2 helper T cell lines and hybridomas, as well as to naive T cells, and to provide accessory cell function for the synthesis of antibody from primed B cells. One of the cell lines expressed MHC class II molecules and was the only line with constitutive antigen-presenting activity for Th1 cells. It may represent a subset of splenic macrophages responsible for the activation of naive Th1 helper cells in situ. The remaining six cell lines responded to INF-gamma by up-regulating their class II expression and acquiring Th1 antigen-presenting activity. They may represent cells which, in situ, lack constitutive antigen-presenting activity but are promoted to presenting status by Th1-derived INF-gamma. Five of the cell lines provided accessory cell function to Th2 cells, as indicated by antibody synthesis in suspensions of spleen cells from primed mice depleted of their antigen-presenting cells. One of the cell lines lacking accessory cell activity had constitutive antigen-presenting activity for Th1 cells. This reciprocal expression of antigen-presenting activity supports the idea that Th1 and Th2 helper cells are activated by different antigen-presenting cells. Finally, the cell lines differed in their ability to constitutively induce an allogeneic response; a response that was limited to CD8+ T cells occurred in a CD4+ helper cell-independent manner and was unaffected by the addition of INF-gamma. The alloantigen-presenting macrophage cell lines also possessed the most efficient accessory cell activity for antibody synthesis. These cell lines, which represent a spectrum of antigen-presenting activities in the spleen afford models for defining the roles of macrophages in the induction of immune responses and for resolving issues concerning their development.     

Variable mutagenic activity of commercial preparations of ABTS (2,2'-azino-di(3-ethyl-benz-thiazoline) sulphonic acid

Different batches of ABTS obtained from the same commercial source varied in their capacity to effect direct mutation in the strains of Salmonella typhimurium used routinely in the incorporation test of Ames. One batch, obtained in 1976, and another obtained early in 1979, both exhibited direct base-pair substitution and frame-shift activities. These activities, however, were absent from each of two batches obtained after 1979, and also from a highly purified preparation from a different source. The possible presence of the unsulphonated immediate precursor of ABTS as a mutagenic impurity is an unlikely explanation for the activity of the mutagenic preparations. It is more probable that the commercial synthesis generated other, mutagenic, impurities which remained in the batches obtained in 1976 and early in 1979, but were absent or were removed from later batches. The identity of these active impurities is unknown. Pure ABTS is neither a direct nor an indirect mutagen.