Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells.

Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.

Bacterial polysaccharides, endotoxins, and immunomodulation.

These studies show that at least some--though certainly not all--of the adjuvant effects of LPS and its derivatives can be attributed to its ability to eliminate the inhibitory effects of Ts which are activated during the course of a normal immune response. The ability of nontoxic MPL to act in this fashion suggests that it can be used as a safe and acceptable alternative to Freund's complete adjuvant to increase the immunogenicity of poorly immunogenic antigens. More important, the ability of MPL to eliminate the expression of Ts activity, without adversely influencing other T cell functions (e.g., Th, Ta, and Tc activity) makes its use as an adjuvant even more promising since it can then permit those T cell functions to be expressed in a much more efficient manner. Obviously, this would have great significance for the development of tumor immunity.

Isolation and purification of cell wall polysaccharide of Bacillus anthracis (delta Sterne).

A polysaccharide fraction was isolated form sodium-dodecyl-sulfate (SDS) treated cell walls of Bacillus anthracis (delta Sterne) by hydrofluoric acid (HF) hydrolysis and ethanolic precipitation. The polysaccharide fraction was subsequently purified by several washings with absolute ethanol. Purity of the isolated polysaccharide was tested using the anthrone assay and amino acid analyzer. The molecular mass of the polysaccharide fraction as determined by gel filtration chromatography was about 12000 Da. Preliminary analyses of the polysaccharide was done using thin layer chromatography and amino acid analyzer, and results obtained from these analyses were further confirmed by gas liquid chromatography and 13C-NMR spectroscopy. Results showed that the polysaccharide moiety contained galactose, N-acetylglucosamine, and N-acetylmannosamine in an approximate molar ratio of 3:2:1. This moiety was devoid of muramic acid, alanine, diaminopimelic acid, glutamic acid, and lipid, thus indicating that the isolated polysaccharide was of pure quality.

Differential effects of monophosphoryl lipid A on expression of suppressor T cell activity in lipopolysaccharide-responsive and lipopolysaccharide-defective strains of C3H mice.

Lipopolysaccharide (LPS)-responsive and LPS-defective strains of C3H mice did not differ in the capacity to make an antibody response to type III pneumococcal polysaccharide or in the degree of thymus-derived suppressor cell (Ts) activity generated following exposure to type III pneumococcal polysaccharide. However, treatment with monophosphoryl lipid A (MPL) abolished the expression of Ts function in LPS-responsive but not LPS-defective mice. Since this effect was elicited by different preparations of MPL, it appears to be a general property of MPL mediated by direct action of MPL on activated Ts.