STUDIES ON THE MODE OF ACTION OF DIPHTHERIA TOXIN : IV. SPECIFICITY OF THE COFACTOR (NAD) REQUIREMENT FOR TOXIN ACTION IN CELL-FREE SYSTEMS

The ability of a number of nucleotides related to NAD to replace NAD as cofactors for inhibition by diphtheria toxin of peptide bond formation has been examined. Neither NADH nor NADP are active. Of some 14 analogues closely related structurally to NAD that have been tested, only 3-thiocarboxamide pyridine-AD is as active as NAD itself. Replacement of the 3-carboxamide group on the pyridine ring by an acetyl group, or deamination of the purine ring, resulted in derivatives with reduced activity. The results were interpreted as suggesting that NAD and certain related nucleotides are capable of specific interaction with diphtheria toxin. Using the method of equilibrium dialysis, reversible binding of 1 mole of NAD per mole of toxin has been demonstrated. Toxoid does not interact with NAD

THE EFFECTS OF DIPHTHERIA TOXIN ON THE CECROPIA SILKWORM

1. The metamorphosis of the Cecropia silkworm is accompanied by large and systematic changes in the insect's sensitivity to diphtheria toxin. 2. Injection of less than 1 gamma of toxin into mature caterpillars, prepupae, or developing adults causes cessation of development followed by delayed death 1 to 5 weeks later. 3. Dormant pupae, on the contrary, are resistant to 70 gamma of toxin and may survive even this enormous dose for over 4 weeks. One-hundredth of this dose, however, prevents pupae from initiating adult development. 4. Tetanus toxin, to which the insect is insensitive, failed to duplicate any of these effects. 5. Maximal sensitivity to diphtheria toxin is characteristic of those stages in the life history which depend on the presence and function of the cytochrome system. Resistance to the toxin, as in the case of the diapausing pupa, is correlated with the existence and utilization of metabolic pathways other than the usual cytochrome system. 6. This correlation persists within the individual insect. Thus, within the diapausing pupa, the toxin fails to affect the heart in which a normal cytochrome system is absent, but, within the same insect, causes a degeneration of the intersegmental muscles in which an intact cytochrome system is present. 7. These several lines of evidence are interpreted in support of the conclusion that diphtheria toxin acts by blocking the synthesis of one or more components in the cytochrome system

A QUANTITATIVE STUDY OF THE SCARLET FEVER TOXIN-ANTITOXIN FLOCCULATION REACTION

1. Highly purified scarlet fever toxin has been prepared from culture filtrates of two scarlatinal strains (NY5 and 594B) of hemolytic streptococcus grown on a medium of defined composition. 2. The flocculation reaction of Rane and Wyman has been studied quantitatively and has been shown specific for scarlet fever toxin and antitoxin. 3. Scarlet fever toxin from strains NY5 and 594B are quantitatively identical in their immunological behavior. 4. Pure scarlet fever toxin contains 0.00023 mg. nitrogen per flocculating unit and close to 1.3 x 108 skin test doses per mg. nitrogen as calculated from the immunological data. Both the immunological and the analytical data suggest that scarlet fever toxin is a protein. Similar calculations indicate that scarlet fever antitoxin contains 0.00093 mg. nitrogen per unit. 5. The scarlet fever toxin-antitoxin complex is readily dissociated in dilute solutions. In this respect the scarlet fever toxin-antitoxin system contrasts sharply with the diphtheria toxin-antitoxin system. 6. The scarlet fever toxin-antitoxin reaction is discussed in relation to other flocculation reactions

STUDIES ON THE MODE OF ACTION OF DIPHTHERIA TOXIN : VI. SITE OF THE ACTION OF TOXIN IN LIVING CELLS

Using the technique of radioautography, it has been shown that a probable maximum of only 25–50 molecules iodine-125-labeled toxin per cell is bound by human HeLa cells treated with approximately 107 molecules of toxin per cell, or just under one saturating dose. Radioautographs of sections from labeled cells locate most if not all of the toxin molecules fixed to the outer cell membrane. Under identical conditions far less label is taken up by mouse L cells. It is probable that the resistance of this species to diphtheria toxin can be accounted for in terms of the failure of mouse cells to bind the toxin protein. The irreversible inhibition of protein synthesis in a living cell culture by a few molecules of toxin located at the cell surface is discussed in relation to the known interaction between toxin, NAD, and transferase II in mammalian cell extracts

DELAYED HYPERSENSITIVITY : III. SPECIFIC DESENSITIZATION OF GUINEA PIGS SENSITIZED TO PROTEIN ANTIGENS

Guinea pigs rendered hypersensitive (delayed-type) to protein antigen can be completely and specifically desensitized by a single injection containing a sufficient amount of the corresponding antigen. Although 1 to 2 mg. of specific antigen are required for complete desensitization, as little as 20 µg. suffices to decrease the size of specific skin reactions in sensitized animals. The duration of non-reactivity lengthens as the amount of antigen in the desensitizing injection is increased, but skin reactivity eventually returns and is accompanied by the appearance of excess circulating antibody. Desensitization can be accomplished with the antigen-antibody complex as well as by "free" antigen. The appearance of delayed skin reactions can be prevented in fully sensitized animals by intravenous desensitization 2 or more hours after intradermal challenge or by simply skin testing with a desensitizing dose of specific antigen. Injection of a desensitizing dose of antigen into specifically sensitized animals also results in a transient anergic state, the implications of which are discussed

EXPERIMENTAL RICKETS IN RATS : I. A DIET PRODUCING RICKETS IN WHITE RATS, AND ITS PREVENTION BY THE ADDITION OF AN INORGANIC SALT.

1. A simple diet is presented which regularly induced rickets in young rats. 2. The substitution of 0.4 per cent secondary potassium phosphate for a small part of calcium lactate in this diet completely inhibited the development of rickets. 3. Quantitative determinations of calcium in the bodies of parallel rats showed a marked increase of calcium content in the rats receiving the added phosphate over those which developed rickets. 4. While it is thus shown by x-rays and by histological examinations and by quantitative chemical analysis that added potassium phosphate increased the assimilation and normal deposition of calcium, it may be the quantitative relationship between the inorganic ions rather than actual deficiency of any one of them which was here the determining factor in the cause or prevention of rickets. Our experiments and conclusions do not exclude the possibility of other causes of rickets than those here discussed

IMMUNOCHEMICAL STUDIES OF ANTITOXIN PRODUCED IN NORMAL AND ALLERGIC INDIVIDUALS HYPERIMMUNIZED WITH DIPHTHERIA TOXOID : II. A COMPARISON BETWEEN THE IMMUNOLOGICAL PROPERTIES OF PRECIPITATING AND NON-PRECIPITATING (SKIN-SENSITIZING) ANTITOXINS

1. The immunological properties of two contrasting types of human antisera, each containing a high titer of diphtheria antitoxin, have been investigated. 2. Sera which contain only non-precipitating antitoxin exhibit most of the properties of atopic reagin-containing sera. This type of antitoxin is capable of sensitizing normal human skin to toxin or toxoid and remains for many weeks in the injected area. It exhibits no Danysz effect, does not fix complement unless very large amounts of serum are used, and can be specifically coprecipitated by addition of precipitating antitoxin and toxin. On the other hand, it is capable of sensitizing guinea pigs to fatal anaphylactic shock. Heating at 56°C. for 4 hours destroys the skin-sensitizing properties and results in almost quantitative conversion to a modified antitoxin which is capable of blocking the wheal and erythema reaction caused by injection of toxoid into sensitized skin. Heating at 56°C. does not result in an appreciable loss of neutralizing power as judged by tests in rabbit and human skin. The anaphylactogenic property of nonprecipitating antibody is likewise unaffected by heat at 56°C. 3. Precipitating antitoxin is incapable of sensitizing normal skin to toxin or toxoid and disappears rapidly from the injected sites. It fixes complement to high titer and sensitizes guinea pigs to fatal anaphylactic shock. It was possible to demonstrate inhibition of the wheal and erythema reaction in sensitized skin by injecting certain mixtures of precipitating antitoxin and toxoid. 4. The two antitoxic sera studied in greatest detail represented extreme cases. Many persons immunized with toxoid developed both precipitating and nonprecipitating antitoxin simultaneously

IMMUNOCHEMICAL STUDIES OF ANTITOXIN PRODUCED IN NORMAL AND ALLERGIC INDIVIDUALS HYPERIMMUNIZED WITH DIPHTHERIA TOXOID : I. RELATIONSHIP OF SKIN SENSITIVITY TO PURIFIED DIPHTHERIA TOXOID TO THE PRESENCE OF CIRCULATING, NON-PRECIPITATING ANTITOXIN

In Vol. 95, No. 4, April 1, 1952, the following sentence should be inserted in the paper by Drs. Kuhns and Pappenheimer on page 365, in the fifth line from the bottom:— The high antitoxin titer found at this time is explained by the fact that the boy had already received an earlier "booster" dose of toxoid a few months previously

STUDIES ON THE MODE OF ACTION OF DIPHTHERIA TOXIN : III. SITE OF TOXIN ACTION IN CELL-FREE EXTRACTS

Extracts from HeLa cells treated with excess diphtheria toxin for several hours, until all protein synthesis has been arrested, are still able to stimulate the poly U-directed incorporation of phenylalanine into polypeptides at a moderate rate. Activity may be restored to normal levels or above by addition of a soluble enzyme fraction containing transferase II. Our results are in agreement with those of Collier who has recently shown that toxin inactivates transferase II in extracts from rabbit reticulocytes. We have further demonstrated that amino acid incorporation in extracts from intoxicated HeLa cells is limited by their transferase II content whereas, in extracts from normal cells, it is the ribosomes and to a lesser extent sRNA that are limiting. We have found that only soluble transferase II is inactivated by toxin; the ribosome-bound enzyme is resistant

TRANSFER OF DELAYED HYPERSENSITIVITY TO DIPHTHERIA TOXIN IN MAN

Simultaneous transfer of delayed hypersensitivity to diphtheria toxin and to tuberculin has been accomplished in eight consecutive instances in man using extracts from washed leucocytes taken from the peripheral blood of tuberculin-positive, Schick-negative donors who were highly sensitive (i.e., pseudoreactors) to purified diphtheria toxin and toxoid. The leucocyte extracts used for transfer contained no detectable antitoxin. The recipient subjects were Schick-positive (<0.001 unit antitoxin per ml. serum) and tuberculin-negative at the time of transfer. All the recipients remained Schick-positive for at least 2 weeks following transfer and in every case their serum contained less than 0.001 units antitoxin at the time when they exhibited maximal skin reactivity to toxoid. Evidence is presented which indicates that the transfer factor may be released from leucocyte suspensions under mild conditions in which most of the cells appear to remain morphologically intact. Four adult Schick-positive subjects have been sensitized to diphtheria toxoid by intradermal injection of a few micrograms of purified toxoid in the form of a washed toxoid-antitoxin precipitate. Two of these sensitized individuals showed severe delayed skin reactions specifically directed against diphtheria toxin (or toxoid) at a time when their serum antitoxin level was less than 0.001 units/ml