Toxins activation with

Bacillus thuringiensis produces a variety of organic compounds which are toxic to the larvae of certain susceptible insect hosts. Among the toxic entities are proteinaceous crystals, probably three soluble toxins, and certain enzymes. The protein material is the major toxin active in killing lepidopterous larvae. The protein is formed by the cells apparently in close synchrony with sporulation, and its nature is a constant function of bacterial strain. The mode of action of the protein is under study. The sequence of events in the pathology observed is probably solubilization of the crystal (enzymatic or physical)—>liberation of toxic unit—>alteration of permeability of larval gut wall— change in hemolymph pH—>invasion of hemolymph by spores or vegetative cells of the bacterium. 

The cellular/molecular mechanism of action for these cyclic peptide toxins is now an area of active research in several laboratories. These peptides cause striking ultrastructural changes in isolated hepatocytes (95) including a decrease in the polymerization of actin. This effect on the cells cytoskeletal system continues to be investigated and recent work indirectly supports the idea that these toxins interact with the cells cytoskeletal system (86,96). Why there is a specificity of these toxins for liver cells is not clear although it has been suggested that the bile uptake system may be at least partly responsible for penetration of the toxin into the cell (92). 

SATA has been used to form conjugates with avidin or steptavidin with excellent retention of activity (Chapter 23, Section 3.1). It also has been used in the formation of a therapeutically useful toxin conjugate with recombinant CD4 (Ghetie et al., 1990), to study syntaxin proteins (Amessou et al., 2007), to prepare bispecific antibodies (Lindorfer et al., 2001), and to make a unique polylysine conjugate as a vehicle for drug delivery (Sakharov et al., 2001). 

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In this case, the antibody is thiolated to contain a sulfhydryl group by modification with SPDP followed by reduction with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect the final conjugate through a disulfide bond.

For instance, if toxin A chain-antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of... 

Figure 21.7 An intact A-B subunit toxin molecule may be activated with 2-iminothiolane with good retention of cytotoxic activity. The thiolated toxin then may be conjugated with SPDP-activated antibody to generate the immunotoxin conjugate through a disulfide bond.

Figure 21.8 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to form a thiol-reactive derivative. Reduction of an A-B toxin molecule with DTT can facilitate subsequent isolation of the A chain containing a free thiol. Mixing the A-chain containing a sulfhydryl group with the SMPT-activated antibody causes immunotoxin formation through disulfide bond linkage. The hindered disulfide of an SMPT crosslink has been found to survive in vivo for longer periods than conjugates formed with SPDP.

Figure 21.11 Fab antibody fragments containing free thiols can be activated with Ellman s reagent to form a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol group may be coupled to the activated Fab molecule to produce an immunotoxin complex.

Figure 21.14 Activation of an intact A-B toxin molecule with MBS with subsequent conjugation with a reduced antibody fragment to produce an immunotoxin.

The toxoid is then prepared by treating the active toxin produced with formaldehyde. The product is normally sold as a sterile aqueous preparation. Tetanus vaccine production follows a similar approach. Clostridium tetani is cultured in appropriate media. The toxin is recovered and inactivated by formaldehyde treatment. Again, it is usually marketed as a sterile aqueous-based product. 

The ultimate localization of LC depends on toxin type, with, for example, type A residing near the plasma membrane and type E remaining in the cytosol, a phenomenon that may explain the differential stability of the toxin isoforms and the persistence of their action. Nevertheless, the LC fragment is always effectively sequestered from the degradative cycle of the terminal. Tyrosines in both LC and HC may be phosphorylated by Src (see Ch. 24), enhancing the stability and proteolytic activity of LC. 

The most common and simplest procedure is to place a few microliters of the test solution over a small puncture wound on a detached leaf. The puncture wound enhances the access of the toxin to the leaf tissue. The leaf is then placed in a petri dish containing a filter paper saturated with water. The top cover of the plate is sealed with parafilm, and the plate is incubated under controlled light and temperature conditions. Toxin activity is usually indicated by chlorotic, necrotic, or colored spots on the leaf. Other methods for bioassay involving CO2 fixation, or effects on organelles, whole plants, protoplasts, tissue cultures, or plant parts are outlined (, 7). 

Sudden infant death syndrome. Water-soluble smoke extract, in cell culture supernatants of mouse fibroblasts (L-929 cell line), produced an increase in TNF-a from respiratory syncytial virus-infected cells. It decreased TNF-a from cells incubated with toxic shock syndrome toxin. Incubation with cigarette smoke extract decreased the NO production from respiratory syncytial virus-infected cells and increased the NO production from cells incubated with toxic shock syndrome toxin. Monocytes from a minority of individuals demonstrated extreme TNF-a responses and/or very high or very low NO. The proportion of samples in which extreme responses with a very high TNF-a and very low NO were detected was increased in the presence of the three agents to 20% compared with 0% observed with toxic shock syndrome toxin. One to 4% was observed with cigarette smoke extract or respiratory syncytial virus L Symphatomimetic activity. Water extract of the dried leaf, administered intravenously to cats at doses of 0.05 and 10-20 mg/kg. 

SATA has been used to form conjugates with avidin or steptavidin with excellent retention of activity (Chapter 13, Section 3.1). It has been used in the formation of a therapeutically useful toxin conjugate with recombinant CD4 (Ghetie etal., 1990A). 

For instance, if toxin A chain—antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of excess cross-linker, the activated antibody can be directly mixed with isolated A chain to create the conjugate (Fig. 320). This procedure makes use of the indigenous sulfhydryl residues produced during reductive separation of the A and B chains and therefore does not require cross-linker thiolation of one of the proteins. 

Figure 322 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to form a thiol-reactive derivative. Reduction of an A—B toxin molecule with DTT can facilitate subsequent...

---