Physicochemical and Biological Properties of Sonically Treated Vi Antigen

Electrophoretically purified Vi antigen from Citrobacter freundii 5396/38 was depolymerized by sonic treatment. The treatment caused an 80% reduction in specific viscosity and a reduction in molecular weight from 1.6 x 10(6) to 3.9 x 10(4). The O-acetyl and N-acetyl contents of the antigen and its infrared spectrum remained unchanged. The sonically treated antigen was only 1% as effective as the original antigen in eliciting protection in mice against challenge with Salmonella typhi. Sonically treated antigen also elicited lower antibody titers after single injections in mice and rabbits. No loss in ability to precipitate antibody or to sensitize red blood cells for hemagglutination was observed.     

THE INTRACELLULAR LOCALIZATION OF PITUITARY THYROTROPIC HORMONE

The intracellular localization of a bovine anterior pituitary preparation of thyroid-stimulating hormone (TSH) was studied in guinea pigs and dogs. The preparation was administered intravascularly or applied directly to tissue sections. TSH was detected by an indirect technique utilizing bovine TSH antiserum and fluorescein-labeled anti-rabbit globulin; the presence of TSH in the tissue was indicated by fluorescence when the tissue was examined under the microscope with an ultraviolet light source. After either intravascular administration or direct application of the TSH preparation, striking fluorescence was found in the nuclei of the thyroid cells and to a lesser degree in the nuclei of retro-orbital fat tissue and kidney tubules in both species studied. A little fluorescence was also seen in spleen tissue. No fluorescence was noted in comparable tissues removed from control animals injected with bovine albumin or globulin or when the tissues were treated with the fluorescein-labeled globulin alone. Fluorescence was also noted in the nuclei of adrenal cells treated with unabsorbed antiserum, but this was greatly diminished when antiserum absorbed with crystalline ACTH was used. The positive reactions were all markedly decreased when the tissues were treated with antisera absorbed with the original TSH preparation. Fluorescence was noted in the cytoplasm of pituitary tissue from both treated and control animals, suggesting a cross-reaction between the bovine pituitary antisera and guinea pig or dog hypophysis. The indirect technique seems to be highly satisfactory for demonstration of the pitiutary hormone within the cell. In addition, the demonstration of immunologically active anterior pituitary TSH bound to cell nuclei offers a clue to the site of action of this hormone.     

The Creatine Phosphoryltransfer Reaction in Iodoacetate-Poisoned Muscle

The iodoacetate-nitrogen-poisoned muscle offers the possibility of studying the stoichiometry of the single muscle twitch since metabolic resynthesis by glycolysis and oxidative phosphorylation are blocked, and there remains as an energy source only the creatine phosphoryltransfer system, creatine phosphate reacting with adenosinediphosphate to give the triphosphate and creatine. It is shown, preparatory to a determination of the amount of phosphocreatine split in a single twitch, that iodoacetate does not inhibit creatine phosphoryltransferase at concentrations which block glycolysis. An analysis is developed which assumes that the transferase maintains the creatine phosphoryl transfer reaction in equilibrium following contraction, and further that the creatine phosporyltransfer reaction and the myokinase reaction are isolated in muscle. On the basis of this analysis and the data obtained, an estimate of the equilibrium constant of the creatine phosphoryl reaction in muscle is obtained which agrees with values determined in vitro. Using the estimated equilibrium constant, and the concentrations of creatine, creatine phosphate, and adenosinetriphosphate found, a value for the concentration of free adenosinediphosphate is obtained which is considerably less than that found by direct chemical analysis.