Treatment with succinic anhydride improves the immunogenicity of Shigella flexneri type 2a O-specific polysaccharide-protein conjugates in mice.

Seroepidemiological data and a clinical trial with a Shigella sonnei O-specific polysaccharide (O-SP)-Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conjugate provide evidence that a critical level of immunoglobulin G (IgG) lipopolysaccharide (LPS) antibodies in serum confers protection against shigellosis. We evaluated the immunogenicity of conjugates whose carrier proteins and O-SPs were treated with succinic anhydride (SA), which reacts with amino groups at neutral pH to form amide-linked carboxyls (succinylation). Conjugates were synthesized with either of two genetically inactivated medically useful toxins, the diphtheria protein CRM9 or rEPA, bound to the O-SP of Shigella flexneri type 2a. Conjugates composed of the succinylated protein, succinylated O-SP, or both succinylated components were administered to mice by a clinically relevant scheme, and their levels of serum IgG anti-LPS and anti-proteins were assayed 7 days after the second and third injections. CRM9 served as a more immunogenic carrier than rEPA. Conjugates composed of succinylated components were more immunogenic than the conjugates composed of the native components. SA treatment of both the carrier protein and the O-SP did not confer an advantage over the succinylated protein alone. Conjugates prepared with native proteins, in general, elicited slightly higher levels of IgG protein antibodies than conjugates composed of the SA-treated proteins.

Increased phosphorylation of specific nuclear proteins in superior cervical ganglia and PC12 cells in response to nerve growth factor.

Treatment of rat superior cervical ganglia in culture with nerve growth factor (NGF) increases the amount of radioactive phosphate incorporated into a nuclear protein band. This band migrates coincidentally with H1 histone on 10% sodium dodecyl sulfate/polyacrylamide gels. The increase in phosphate incorporation is at least 70% and occurs only in tissues known to be responsive to NGF. It is not produced by treatment with related peptides, but is observed after the addition of dibutyryl cyclic AMP. An increase in phosphorylation can be detected after 1 h, and can be seen with as little as 10 ng/ml of NGF in the medium. Neither actinomycin D nor cycloheximide inhibits the effect. When the nuclei are extracted with 0.2 M H2SO4 and the extract analyzed on acid-urea/polyacrylamide gels, two NGF-responsive proteins can be detected. One protein again migrates with the H1 histone marker; the other migrates more slowly than H1. These two NGF-responsive proteins have molecular weights of approximately 30,000 and are chromatin-bound. They are not soluble in 5% perchloric acid, but can be extracted from the nuclei with 0.35 M NaCl. No increase in the phosphorylation of these proteins was seen in ganglia from 6-hydroxydopamine-treated rats. The phosphorylation of the proteins in both control and NGF-treated ganglia occurs almost exclusively on serine residues. The amino acid compositions of the two nuclear proteins show that they are different from the H1 histone and different from each other. Both nerve growth factor (NGF) and epidermal growth factor (EGF) increase the incorporation of radioactive phosphate into a specific nuclear protein in cultures of PC12, a clone of rat pheochromocytoma. Purified NGF antibody blocks the effect of NGF, but not that of EGF; EGF antiserum neutralizes the effect of EGF, but not that of NGF. Insulin, glucagon, and dexamethasone are without effect. The increase in phosphorylation due to NGF can be detected within 1 h. Dibutyryl cyclic AMP increases the phosphorylation of this protein, but dibutyryl cyclic GMP does not. Neither the uptake nor the overall incorporation of [32P]orthophosphate is altered by NGF, EGF, or dibutyryl cAMP under the present experimental conditions. The nuclear protein exhibiting increased radioactivity is similar in solubility, size, and amino acid composition to one of the NGF-responsive nuclear proteins from sympathetic ganglia.