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T B Helting

Publications and source records attributed to T B Helting.

12 recordsLinked to original sources
PubMed1984

Analysis of the immune response to papain digestion products of tetanus toxin.

Tetanus toxin was degraded by papain and the previously characterized Fragment B and Fragment C were isolated. The induction of protective immunity against tetanus toxin was subsequently investigated in two animal models using Fragment B and Fragment C, as well as conventional tetanus toxoid for comparison. In guinea pigs, Fragment B showed an immunizing potency similar to that recorded for an equal amount of tetanus toxoid, whereas Fragment C was considerably less efficient, on an equal weight basis. In contrast, mice immunized with Fragment C reached a protection level similar to that achieved after immunization with the complete tetanus toxoid antigen. In both animal models, Fragments B and C acted in a synergistic fashion when injected together. Analysis of sera from guinea pigs using ELISA, after administration of a low dose of tetanus immunogen corroborated these results. The data indicate that antigenic determinants in distinct regions of the toxin molecule independently may induce antibodies neutralizing the lethal action of tetanus toxin. The immune response to the complete toxoid antigen may be subject to influence by intramolecular competition between antigenic determinants. The immunogenicity of the fragments as measured by the protection rate after active immunization may depend upon the animal species used.

Animals↗
PubMed1981

Structure of tetanus toxin: the arrangement of papain digestion products within the heavy chain-light chain framework of extracellular toxin.

Upon digestion with papain, single chain intracellular tetanus toxin was completely converted to the extracellular form of the toxin, which consists of two disulfide-linked polypeptide chains (heavy chain and light chain of tetanus toxin). A portion of the material was degraded further by papain to yield the two major protein fragments, B and C, respectively, previously identified after digesting extracellular tetanus toxin with this enzyme. Thus it may be concluded that the order of release of these of these fragments from the intracellular toxin does not provide a clue as to their position within the original molecule. However, N-terminal analysis of the two fragments in conjunction with recent N-terminal data on tetanus toxin itself clearly indicated that fragment B of tetanus toxin contains the light chain polypeptide and the N-terminal portion of the heavy chain, whereas fragment C is derived from the C-terminal portion of the heavy chain.

Clostridium tetani↗
PubMed1981

Extracted protective antigen of Bordetella pertussis. I. Preparation and properties of the solubilized surface of components.

Bordetella pertussis microorganisms were treated with several extracting agents followed by ultracentrifugation to remove particulate matter. Analysis of the resulting supernatants by SDS gel electrophoresis showed one major component after simple salt extraction, and much more complex, although consistent pattern following detergent treatment. The yield of the solubilized protein in detergent extracts exceeded by far the values recorded for salt extracts. In order to prevent irreversible precipitation of the solubilized proteins upon removal of the denaturing agent, a novel procedure was developed. After extraction with urea-salt, the solubilized material was absorbed on a mineral carrier prior to the separation of the denaturing agent. The resulting absorbed vaccine was highly potent in the mouse-protection test, whereas the toxic reactions, elicited upon injection into experimental animals, were reduced in the comparison to the starting material. This diminished reactogenic potential was accompanied by the partial loss of the leukocytosis-promiting factor, whose activity was greatly diminished by urea-salt at alkaline pH-values. The procedure described may be applied to large-scale processing of Bordetella persussis microorganisms. Clinical trials now in progress should confirm or rebut the thesis that increased tolerability of the product, inferred from animal experiments, is reflected by fewer adverse reactions in humans. In the former case, the detergent extract vaccine may constitute a realistic alternative to conventional whole-cell vaccines against whooping-cough.

Absorption↗
PubMed1981

Serotype determinant protein of Neisseria Meningitidis. Large scale preparation by direct detergent treatment of the bacterial cells.

Neisseria meningitidis Group B microorganisms, inactivated with phenol and harvested by centrifugation, were subjected to direct treatment with various detergents to solubilize the serotype determinant proteins localized in the outer membrane. Analysis of the data showed that extraction of the cells with detergents provided yields of the serotype protein substantially exceeding those obtained by simple salt extraction of the bacteria. Routinely, more than 2 mg of end product per g of cell mass (wet weight) may be recovered by the present method. By gel chromatographic analysis, the serotype determinant protein was shown to interact with the capsular polysaccharides derived from Group A or C Neisseria meningitidis microorganisms, forming high molecular weight complexes. This interaction markedly enhanced the solubility of the serotype determinant protein. Combined vaccines of the type-specific protein with the group- specific polysaccharides were evaluated for their immunogenic potential in the subcutaneous steel spring implant model. In guinea pigs, amounts corresponding to 10 micrograms completely prevented infection upon challenge with homologous organisms four weeks after immunization. Partial protection was observed with immunizing doses corresponding to 2 micrograms or 0.4 micrograms/animal, respectively. Compared to lyophilized preparations, vaccines adsorbed to a mineral carrier were slightly less effective in inducing protection, whereas inclusion of Bordetella pertussis as a component of the vaccine stimulated the immune response.

Animals↗
PubMed1979

Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form.

Protease activity has been demonstrated in culture supernatants of Clostridium tetani at various stages of fermentation. Gel chromatography of the concentrated filtrates revealed the presence of three enzymatically active fractions eluting at separate positions off the column. The smallest protease was found to "nick" the single chain intracellular tetanus toxin, producing the extracellular, two-chain structure of the molecule. As little as 3 ng of active protease were sufficient to cleave 50 microgram of intracellular tetanus toxin, suggesting that this enzyme is responsible for the observed structural change of the toxin molecule during its release into the culture medium. By comparison, the second protease, eluting at an intermediate position, exhibited only marginal activity towards intracellular toxin. The third, largest, enzyme was not active under the conditions of the assay. However, the latter protease effectively hydrolyzed low molecular weight histidyl peptides, and it is concluded that this enzyme is similar to the one described by Miller, P.A. Gray, C.T., and Eaton, M.D. (1960) J. Bacteriol. 79, 95-102. The properties of the partially purified enzymes, including their differential behavior towards a number of protease inhibitors, are reported.

Clostridium tetani↗
PubMed1977

Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments.

Tetanus toxin was digested with papain, yielding one major polypeptide (Fragment C) with a molecular weight corresponding to 47,000 +/- 5%, thus comprising about one-third of the toxin molecule. Fragment C was antigenically active, atoxic, and stimulated the formation of antibodies neutralizing the lethal action of tetanus toxin in vivo. Furthermore, a second split product (Fragment B) was isolated from the papain digest, containing two polypeptide chains linked together via a disulfide bond. Fragment B (Mr = 95,000 +/- 5%) was atoxic and showed a reaction of nonidentity with Fragment C on immunodiffusion analysis against tetanus antitoxin. The basic two-chain structure (heavy and light chain polypeptide, cf. Matsuda, M., and Yoneda, M. (1975) Infect. Immun. 12, 1147-1153) of tetanus toxin has been confirmed and the relationship between Fragments B and C within this framework has been established. Fragment C was distinguished from the light chain by electrophoresis in sodium dodecyl sulfate and by immunodiffusion analysis, indicating that this fragment constitutes a portion of the heavy chain polypeptide. Fragment B showed a reaction of partial identity with the light as well as the heavy chain from tetanus toxin. Reduction of Fragment B with dithiothreitol followed by gel chromatography yielded a fraction which was indistinguishable from the light chain portion of the toxin molecule. It is concluded that Fragment B comprises the complementary portion of the heavy chain (remaining after scission of the polypeptide bond(s) releasing Fragment C) linked to the light chain by a disulfide bond.

Amino Acids↗
PubMed1977

Structure of tetanus toxin. II. Toxin binding to ganglioside.

The interaction between tetanus toxin and ganglioside containing 2 N-acetylneuraminic acid residues linked in sequence to one another has been investigated using a new method involving radioactively labeled ganglioside and tetanus toxin adsorbed to Sephadex matrix. Binding between the two components was demonstrated, and it was calculated that in the nanomolar concentration range, tetanus toxin becomes half-saturated at about 5 X 10(-8) M concentration of ganglioside. Removal of the ceramide portion from the ganglioside resulted in the complete loss of binding activity, whereas removal of the terminal N-acetylneuraminic acid residue from the intact ganglioside had no effect. Among the fragments derived from tetanus toxin (Helting, T. B., and Zwisler, O. (1977) J. Biol. Chem. 252, 187-193), only the heavy chain polypeptide exhibited a binding activity of the same order of magnitude as that observed for the native toxin. The light chain polypeptide showed no interaction with ganglioside and among the fragments derived from the toxin by digestion with papain, only Fragment C, at a high protein concentration, displayed marginal binding activity. Using monovalent antibodies directed against specific regions of the tetanus toxin molecule, it was demonstrated that antibodies directed against Fragment C uniquely interfere with the binding process. Anti-light chain serum was ineffective, as well as antitetanus toxoid serum previously absorbed with Fragment C. It is concluded that the binding site for ganglioside is located on the heavy chain portion of tetanus toxin, possibly in or near the region comprised by Fragment C.

Binding Sites↗