Kunihiro Ozutsumi

Primary action of Clostridium perfringens type a enterotoxin on hela and vero cells in the absence of extracellular calcium: rapid and characteristic changes in membrane permeability

Clostridium perfringens type A enterotoxin bound rapidly to HeLa and Vero cells in the absence of extracellular Ca2+ at 37 degrees C. The bound toxin rapidly (within 2 min) caused influx of Na+ and efflux of K+ and Mg2+. Changes in membrane permeability occurred in the absence or presence of extracellular Ca2+ and to the similar extents at 37 degrees C and 4 degrees C, in contrast to the subsequent bleb and balloon formation, which required both extracellular Ca2+ and incubation at 37 degrees C. Substances with molecular weights of over ca. 200 protected the cells from the morphological alterations induced by the toxin, whereas substances with molecular weights of less than ca. 200 did not. The mechanism of the primary action of the enterotoxin is discussed.

Acute botulinum-like intoxication by tetanus neurotoxin in mice

Abstract Intravenous injection of purified tetanus toxin(1000-0.06 μg) killed mice within minutes(20–450 min), causing flaccid paralysis indistinguishable from that in botulinum intoxication: a linear relation was found between the log of the toxin dose and that of death time(survival time). The dose and route dependences of the manifestations of the spastic paralysis typical of classical tetanus and of the acute botulinum-like flaccid paralysis were studied in relation to the death time. Treatment of the toxin with trypsin or gangliosides did not affect its acute botulinum-like toxicity. Theophylline delayed the time of acute death due to the botulinum-like intoxication in mice caused by tetanus toxin and provided some protection.

Establishment of stable mouse/human-human hybrid cell lines producing large amounts of anti-tetanus human monoclonal antibodies with high neutralizing activity

To establish stable hybrid cell lines producing human anti-tetanus antibody with high toxinneutralizing activity, peripheral lymphocytes from humans hyperimmunized with tetanus toxoid were, after in vitro antigen stimulation, fused with a mouse/human heteromyeloma or human lymphoblastoid cell line and cloned. Unlike the IgM secretors (six clones), the IgG secretors we obtained (six clones) produced anti-tetanus human monoclonal antibodies with high neutralizing activity (the highest one, cell line G2, 4.3 IU/100 μg IgG).Appropriate combinations of three or four kinds of monoclonal antibodies of the IgG type resulted in markedly increased neutralizing activity comparable with that of anti-tetanus human polyclonal immunoglobulin preparations currently used clinically on the basis of toxin-specific IgG content. Five of these cell lines produced 10≈20 pg of antibody per ml for more than 3 months. The cell line G2 produced 6 mg of antibody per day in serum-free medium in a 500-ml bioreactor in perfusion culture and 13–104 mg in a nude mouse. These cell lines satisfied, for the first time, the minimal requirements for applying human monoclonal antibodies to clinical use.

Enterotoxin of Clostridium perfringens type a forms ion-permeable channels in a lipid bilayer membrane

The enterotoxin of Clostridium perfringens type A was found to form ion-permeable channels in a lipid bilayer. A patch clamp technique was used to detect channel activities in an asolectin bilayer with incorporated enterotoxin. About 20% of the lipid bilayer patches examined showed rectangular or stepwise shift of membrane current. The shifts indicated the gating of ion-permeable channels in the patches. The channels showed high conductance (40-450 pS), no rectification in current-voltage curves and occasional long-lasting events. The significance of these findings is discussed in relation to the mechanism of action of the toxin.

Morphological alterations and changes in cellular cations induced by Clostridium perfringens type a enterotoxin in tissue culture cells

The morphological alterations (bleb-balloon formation) induced by Clostridium perfringens type A enterotoxin in HeLa and Vero cells were studied under defined extracellular conditions. The action of enterotoxin was found to depend on the temperature but not on energy metabolism. The morphological alterations by the enterotoxin occurred in phosphate buffered saline containing Ca2+ and Mg2+. Of the constituents of the buffered saline, Ca2+ was essential for the morphological alterations and other ions were interchangeable. The morphological alterations by the enterotoxin occurred also in 10 mM Hepes-Na buffer, pH 7.2 containing NaCl, KCl or choline chloride at a concentration of over ca. 50 mM and in 10 mM Hepes-Ca buffer, pH 7.2 containing CaCl2 at a concentration of over ca. 50 mM. Addition of sucrose to the medium prevented induction of the morphological alterations. The amount of sucrose necessary to protect the cells increased with increase in NaCl, KCl or CaCl2 concentration in the medium. A calcium ionophore A23187 mimicked the action of enterotoxin. Examination of the cation contents of the cells by atomic absorption spectrophotometry showed early and rapid increase of Ca 2+ during intoxication with concomitant changes in Na+, K+ and Mg2+ that reduced the ion concentration gradients between inside and outside of the cell present before toxin treatment. The mechanism of action of C. perfringens type A enterotoxin is discussed on the basis of these findings.

Purification by high performance liquid chromatography of Clostridium perfringens type a enterotoxin prepared from high toxin producers selected by a toxin-antitoxin halo

High enterotoxin-producing substrains of Clostridium perfringens type A were selected reproducibly as colonies having toxin-antitoxin haloes on agar plates of Duncan-Strong medium containing antitoxin serum. Enterotoxin from these sub strains was subjected to rapid purification by high performance liquid chromatography (HPLC). For this, the toxin was extracted by sonication from sporulating bacteria grown in Duncan-Strong sporulation medium, fractionated by ammonium sulfate (40% saturation) precipitation and differential solubilization and then purified by HPLC: gel permeation chromatography through a G2000SW column and ion-exchange chromatography on a Mono Q column. Purified toxin preparations had a similar specific activity (4.2 x 102 mouse MLD/mg protein) and homogeneity on polyacrylamide gel-electrophoresis to preparations obtained by conventional gel permeation through a Sephadex-G200 column. By further HPLC on a Mono Q column, minor nontoxin proteins were separated from the toxin without loss of the toxicity on a protein basis. The final yield of the purified toxin was about 15° of that in the bacterial extract. The two HPLC procedures each took only one hour.

Tetanus toxin: a rapid and selective blockade of the calcium, but not sodium, component of action potentials in cultured neuroblastoma N1E-115 cells.

The slow action potential of mouse neuroblastoma N1E-115 cells, which was elicited in Na-free medium and whose amplitude was directly dependent on extracellular Ca concentrations, was blocked by tetanus toxin (TNTX) without change in resting membrane potential and resistance. The average value of action potential overshoot (peak potential level of spikes) in the absence and presence of TNTX was -1 and -11 mV, respectively. The concentration of TNTX required for 50% inhibition of Ca-dependent spikes (IC50) was about 65 ng/ml. The amplitude of spikes was decreased within 10-20 min after adding TNTX. The inhibition of Ca-dependent spikes was mimicked by 10 mM CoCl2 and MnCl2, but not by the boiled or neutralized toxin. The results indicate that TNTX inhibited electrogenesis of Ca, but not Na, components of action potentials of the neuroblastoma cells.

Tetanus toxin blocks Ca spikes in neuroblastoma clone N1E-115 cells.

The effect of tetanus toxin on the Ca spike of the action potential evoked in neuroblastoma clone N1E-115 cells by constant current stimulation in Na+-free medium containing various concentrations of Ca2+(1.8-80 mM) was examined by intracellular recordings. Tetanus toxin (more than 140 ng/ml) blocked the Ca spikes. The resting membrane potentials and membrane input resistances of tetanus toxin-treated cells were similar to those of untreated cells. The blocking effect of tetanus toxin was evident in medium containing Ca2+ at concentrations of more than 30 mM. The difference between the peak levels of the action potentials of toxin-treated and untreated cells was statistically significant at Ca2+ concentrations of 7.2 mM and more.

Clostridium perfringens type A enterotoxin induces release of noradrenaline from the neurosecretory PC12 cell line.

Clostridium perfringens type A enterotoxin(500 ng/ml) induced extensive release of noradrenaline (1/3-2/3 of the total cell content) from PC12 cells in 2-4 min in the presence, but not the absence of extracellular Ca2+. Cells treated with toxin in the absence of Ca2+ released noradrenaline promptly on subsequent addition of Ca2+ to the medium. The amount of noradrenaline released depended on the concentrations of both Ca2+ and toxin in the medium (ED50, 0.3 mM and 420 ng/ml respectively). Ca2+ could be replaced by Ba2+ or Sr2+, and Mn2+ or Co2+, which are Ca2+ channel blockers, did not inhibit the release of the transmitter. These findings are discussed in relation to the systemic effects of enterotoxin.

Rapid method for purification of Clostridium botulinuh type C neurotoxin by High Performance Liquid Chromatography (HPLC )

The culture supernatant of Clostridium botulinum type C, concentrated by addition of RNA, acid precipitation and subsequent protamine treatment was used as starting material for rapid purification of L toxin (mol. wt. ca. 500K) and M toxin (mol. wt. ca. 350K) of C1 neurotoxin by ion-exchange chromatography on a Mono S column by fast performance liquid chromatography (FPLC). L and M toxins were highly purified further by gel permeation chromatography through a TSK G3000SW column at pH 6.0 by high performance liquid chromatography (HPLC). Purified S toxin (mol. wt. ca. 150K, Cl neurotoxin without a nontoxic component) was then obtained from L toxin rapidly by gel permeation chromatography at pH 7.3 through a TSK G3000SW column by HPLC. Purified S toxin was also obtained rapidly from M and L toxins by ion-exchange chromatography on a Mono Q column at pH 8.0 using an FPLC system. The purified preparations of L, M and S toxins gave single bands on conventional polyacrylamide gel electrophoresis, and had specific activities of 2.8, 6.7, and 14–21 × 107 LD50/mg N, respectively, in mice. On immunoelectrophoresis, purified S toxin gave a single arc against anti-crude toxin serum. The yield of toxicity as L and M toxins was 73.1% (32.5% as L toxin and 40.6% as M toxin) from the protamine-treated concentrated culture supernatant. The recovery of toxicity as S toxin from purified L or M toxin was almost 100% (97.6–100% of L toxin and 97.5% of M toxin). These procedures provide a rapid method for purifying L and M toxins, which have stable toxicities. The method will also be very useful for rapid preparation of the toxic component (S toxin) of C1 neurotoxin, which is unstable, in small amounts from the L and M toxins just before its use in experiments.