Botulinum C2 toxin potentiates activation of the neutrophil oxidase. Further evidence of a role for actin polymerization.

Botulinum C2 toxin was employed as a specific inhibitor of actin polymerization in rat neutrophils to determine its role in oxidase activation. This toxin was shown to inhibit actin polymerization and the microfilament-dependent function, phagocytosis. Oxidase activation in response to the chemotactic peptide, f-Met-Leu-Phe (FMLP) was enhanced approx. 3-fold. The enhancement by C2 toxin did not occur in cells pretreated with cytochalasin B. C2 toxin had no significant effect on the FMLP-induced intracellular Ca2+ rise. These data are consistent with an inhibitory role for actin polymerization in oxidase activation.

Inhibition of phagocytosis by rainbow trout (Oncorhynchus mykiss) macrophages by botulinum C2 toxin and its trypsinized component II.

Botulinum C2 toxin (C2T) is composed of two nonlinked protein components, components I and II. The toxin reconstituted with component I and trypsinized component II inhibited phagocytosis of rainbow trout (Oncorhynchus mykiss) and mouse macrophages. Inhibition in both cell types was observed over a range of toxin concentrations that were not toxic to the cells. Because cytoplasmic action of rainbow trout macrophages is ADP-ribosylated by component I, the inhibition of phagocytosis in trout cells by C2T is probably due to inactivation of cytoplasmic actin. Moreover, phagocytosis by trout cells was also inhibited in a dose-dependent manner by trypsinized component II alone, and did not cause cell death. The present results show that the macrophages of aquatic vertebrates are susceptible to C2T, and that trypsinized component II elicits a novel biological activity by binding to the cell membrane of the macrophages.

NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin.

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two separate protein components, designated components I and II, which individually have little activity, but, when mixed and treated with trypsin, exert the potent activity. The present study provides the evidence that component I of the toxin catalyzes the hydrolysis of NAD into nicotinamide and ADP-ribose, whereas component II does not, indicating that component I of C2T has NAD-glycohydrolase activity, which ability is shared with cholera and diphtheria toxins. However, C2T affected neither glycerol production of fat cells nor protein synthesis in cell-free system. Component I of C2T in the presence of [alpha-32P]NAD radiolabeled a protein of Mr 46,000 in the supernatant fractions of mouse tissue homogenates; the protein was abundant in brain, lung and intestine, whereas there was little or none of the protein in muscle. These results indicate that component I can catalyze the covalent attachment of the ADP-ribose moiety of NAD to intracellular protein, which differs from those modified with cholera and diphtheria toxins. The present data, together with previous findings, suggest that the biological activity of C2T is elicited by ADP-ribosylation activity of component I, which is internalized into the cells after binding to the receptor site introduced with the binding of component II to the cell surface membrane.

ADP-ribosylation of nonmuscle actin by component I of botulinum C2 toxin inactivates the ability to interact with unmodified actin.

Botulinum C2 toxin, elaborated by Clostridium botulinum types C and D, is composed of two dissimilar unassociated proteins, designated components I and II. Component I catalyzes ADP-ribosylation of nonmuscle beta- and gamma-actins but not of muscle alpha-actin. The maximal levels of ADP-ribosylation of the actin were about 1.0 mol of ADP-ribose/mol of actin. Sedimentation velocity analysis showed that ADP-ribosylated actin remained in a monomeric state even under polymerization conditions. In addition to the inactivation of self-polymerization ability, the ADP-ribosylated actin affected neither the initial rate nor the final extent of polymerization of unmodified actin as monitored by the increase in fluorescence intensity of N-pyrenyliodoacetamide-labeled actin. Electron microscopy revealed that no filaments or particles were formed from ADP-ribosylated actin in the conditions favorable for polymerization of unmodified actin; moreover, actin filaments produced from unmodified actin in the presence of ADP-ribosylated actin were not distinguishable from those from unmodified actin alone. These results indicate that the introduction of one ADP-ribose residue into the beta/gamma-actin molecule by component I inactivated the actin, preventing not only the self-assembly of the modified actins but also the interaction with unmodified actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of Clostridium botulinum neurotoxin to gangliosides.

The binding characteristics of Clostridium botulinum neurotoxins of types B, C1, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) or high (150 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) ionic strengths and at 0 or 37 degrees C. The three types of toxins bound exclusively to three kinds of gangliosides, GD1a, GD1b, and GT1b, in both the thin layer chromatography plate and the microtiter plate methods. Type C1 toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37 degrees C. At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4 X 10(8) M-1. In contrast, the binding kinetics of type C1 toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system. The heavy chain facilitated the binding of the toxin to the gangliosides. These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins. The gangliosides may bind to type C1 toxin together with other potential receptor(s) on synaptosomal membranes.

The gene for component-II of botulinum C2 toxin.

The gene encoding component-II of the Clostridium botulinum C2 toxin was cloned from the chromosomal DNA of C. botulinum type C strain (C)-203U28, and the nucleotide sequence was determined. The gene (bc2h) encodes a protein with 721 amino acid residues and is located at 247 bp downstream of the gene for component-I. The N-terminal 16 amino acids were identical to those obtained by analysis of the purified component-II toxin. The ORF for bc2h had only 39% homology at the amino acid level with the C.perfringens iota-Ib protein and an ATP/GTP binding site which is present in the iota-Ib protein is missing from the protein encoded by bc2h. Both genes had a homologous region that predicts a transmembrane segment.

Long-term results of double-door laminoplasty for cervical stenotic myelopathy.

STUDY DESIGN: A retrospective study of the long-term results from double-door laminoplasty (Kurokawa's method) for patients with myelopathy caused by ossification of the posterior longitudinal ligament and cervical spondylosis was performed. OBJECTIVE: To know whether the short-term results from double-door laminoplasty were maintained over a 10-year period and, if not, the cause of late deterioration. SUMMARY OF BACKGROUND DATA: There are few long-term follow-up studies on the outcome of laminoplasty for cervical stenotic myelopathy. METHODS: In this study, 35 patients with cervical myelopathy caused by ossification of the posterior longitudinal ligament in the cervical spine and 25 patients with cervical spondylotic myelopathy, including 5 patients with athetoid cerebral palsy, underwent double-door laminoplasty from 1980 through 1988 and were followed over the next 10 years. The average follow-up period was 153 months (range, 120-200 months) in patients with ossification of the posterior longitudinal ligament and 156 months (range, 121-218 months) in patients with cervical spondylotic myelopathy. Neurologic deficits before and after surgery were assessed using a scoring system proposed by the Japanese Orthopedic Association (JOA score). Patients who showed late deterioration received further examination including computed tomography scan and magnetic resonance imaging of the cervical spine. RESULTS: In 32 of the patients with ossification of the posterior longitudinal ligament and 23 of the patients with cervical spondylotic myelopathy, myelopathy improved after surgery. The improvement of Japanese Orthopedic Association scores was maintained up to the final follow-up assessment in 26 of the patients with ossification of the posterior longitudinal ligament and 21 of the patients with cervical spondylotic myelopathy. Late neurologic deterioration occurred in 10 of the patients with ossification of the posterior longitudinal ligament an average of 8 years after surgery, and in 4 of the patients with cervical spondylotic myelopathy, including the 3 patients with athetoid cerebral palsy, an average of 11 years after surgery. The main causes of deterioration in patients with ossification of the posterior longitudinal ligament were a minor trauma in patients with residual cervical cord compression caused by ossification of the posterior longitudinal ligament and thoracic myelopathy resulting from ossification of the yellow ligament in the thoracic spine. CONCLUSIONS: The short-term results of laminoplasty for cervical stenotic myelopathy were maintained over 10years in 78% of the patients with ossification of the posterior longitudinal ligament, and in most of the patients with cervical spondylotic myelopathy, except those with athetoid cerebral palsy. Double-door laminoplasty is a reliable procedure for individuals with cervical stenotic myelopathy.

Serum hemolytic activity in dogs infected with Babesia gibsoni.

The hemolytic activity in serum of Babesia gibsoni-infected dogs was examined. When the activity was assayed in a reaction system consisting of similar concentrations of the serum and canine red blood cells to those in blood, significant hemolysis was observed. The activity of the serum of B. gibsoni-infected dogs, either naturally or experimentally, was always higher than that of uninfected animals. Moreover, in the experimental infection with B. gibsoni, the change in serum hemolytic activity was parallel to those of anemia and parasitemia, whereas it was inversely parallel to that of the hematocrit value. The present study revealed the presence of a hemolytic factor(s) in the serum of B. gibsoni-infected dogs, suggesting that the progressive anemia was due to hemolysis by the factor(s).

Immunological treatment of cough occurring in dogs with dirofilariosis.

A persistent, spasmic and productive cough known as filarial cough often occurs in dogs with dirofilariosis, and has been considered to be the consequence of an allergic response to Dirofilaria immitis. Twenty-one dogs with filarial cough were subcutaneously injected with worm antigen (200 micrograms of protein concentration) extracted from adult D. immitis once a day for 5 days. These injections were effective for 17 (81%) of the dogs, resulting in a complete cure for 7 dogs and marked improvement for 10 dogs.

CDNA cloning of feline leptin and its mRNA expression in adipose tissue.

Leptin, the product of the obese (ob) gene, is an adipocyte-derived hormone involved in regulating food intake and energy expenditure in humans and rodents. To determine the primary structure of feline leptin, we cloned the feline leptin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of complementary DNA (cDNA) ends (RACE) methods. The full-length feline leptin cDNA was 2935 bp with a 501 bp open reading frame encoding the precursor peptide of 167 amino acids including 21 residues of signal peptide. The sequence of a 146-amino acid mature leptin was 81.5-91.8% homologous to those of other species. RT-PCR analysis revealed that the leptin mRNA was expressed in adipose tissues and not detected in liver, heart, kidney, lung, pancreas. brain and skeletal muscle. These data show that feline leptin is highly homologous to leptins of other species, and expressed in adipose tissues in cats.

Serum hemolytic activity of Babesia gibsoni-infected dogs: the difference in the activity between self and nonself red blood cells.

The serum hemolytic activity of Babesia gibsoni-infected dogs varied when assayed with nonself red blood cells from different dogs, whereas it did not vary when assayed with red blood cells, irrespective of self or nonself, from a particular dog throughout the experiment. The variety in activity determined with nonself red blood cells was not related to the type of red blood cell by DEA, D and J systems. Serum hemolytic activity with self red blood cells was different in the course of infection from that with nonself red blood cells, especially in the late stage of infection, when the activity with self red blood cells decreased more rapidly than that with nonself red blood cells. The results indicate that the serum hemolytic activity of B. gibsoni-infected dogs determined with self red blood cells probably reflects the in vivo activity, suggesting that the rapid decrease in activity in the late stage of infection is a way of acquired resistances for the host to recover from hemolytic anemia in the infection. The facts that the hemolytic activity increased by heating the serum at 56 degrees C, that the osmotic fragility of red blood cells remained almost on the same during the course of infection and that Coombs' test for red blood cells of the infected animal was negative suggest that the immune-mediated hemolytic anemia is not a possible mechanism for the progressive and severe anemia in B. gibsoni-infection. The present results support the previous notion that the increased serum hemolytic activity is at least one of the causes of anemia in canine B. gibsoni-infection.

Antibodies to Clostridium botulinum toxins in free-living birds and mammals.

Naturally-occuring antibodies against Clostridium botulinum toxins were found in Cathartes aura (turkey vultures), Canis latrans (coyotes) and Corvus brachyrhynchos (crows) by the passive hemagglutination (PHA) test and verified by the serum neutralization (SN) test. The prevalence of IHA antibodies was 18 of 20 vultures (90%), 5 of 12 crows (42%) and 25 to 110 coyotes (23%). Vultures and coyotes were seropositive by the PHA test against A, B, C, D, and F toxins. The highest antibody titer 1:8192 was in vulture serum against type C. In descending order, the highest antibody levels were against type C, D, F, E, A and B toxins.

Serologic survey for certain zoonotic diseases in black bears in California.

Black bears (Ursus americanus) from 3 geographic areas of California were tested for antibodies against agents of 6 zoonotic diseases: toxoplasmosis (indirect hemagglutination), Q fever (microagglutination), trichinosis (latex particle agglutination), botulism (passive hemagglutination), leptospirosis (plate agglutination), and plague (enzyme-linked immunosorbent assay). Of 149 sera tested, 40 (27%) were positive for Toxoplasma gondii antibodies and 25 (17%) had antibodies against Coxiella burnetii. Of 141 bears tested for Trichinella spiralis, 18 (13%) were seropositive, and 19 (15%) of 125 tested had antibodies against the plague organism, Yersinia pestis. Only 2% (2 of 123 tested) had antibodies against Clostridium botulinum. Sera from 129 bears were tested against 4 pools of Leptospira interrogans representing 12 serovars, and 16% of the sera reacted with the pool containing the serovars australis, hyos, and mini georgia.

Activation of botulinum C2 toxin by trypsin.

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two dissimilar protein components, designated components I and II. The biological activity of C2T is enhanced by treating the toxin with trypsin. This activation of C2T is observed as a result of mixing untrypsinized component I and trypsinized component II but not as a result of mixing trypsinized component I and untrypsinized component II. The data presented here show that the maximum lethality of C2T, determined by mixing untrypsinized component I and trypsinized component II, was attained by treating component II with trypsin at a ratio of 10:1 on a protein basis for 30 min at 35 degrees C at pH 7.5. The activation of component II was always accompanied by a change in the molecular weight of the component from 101,000 to 88,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the gel filtration of trypsinized component II resulted in the separation of two active components, with apparent molecular weights, estimated from the elution volume by gel filtration, of 365,000 and 74,000. The high-molecular-weight component II had hemagglutination and hemolytic activities, whereas the low-molecular-weight component II has only hemagglutination activity. These two molecular species of active component II had approximately the same lethality, when mixed with component I, and gave a single band in SDS-PAGE, with a molecular weight of 88,000, the same as that of trypsin-activated component II under different reaction conditions. The results indicate that the activation of C2T by trypsin is due to the molecular conversion of component II from molecular weight 101,000 to 88,000 as determined by SDS-PAGE and that the trypsin-activated component II tends to form an oligomer of the active component II.

Response of mouse intestinal loop to botulinum C2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components.

Botulinum C2 toxin, which is composed of two nonlinked protein components, components I and II, induced fluid accumulation in mouse intestinal loops. The secretory response to C2 toxin was initiated after a lag period of 1 to 2 h and increased gradually for at least 10 h. The activity of C2 toxin was enhanced by treatment with trypsin and abolished by neutralization with anti-component I or anti-component II sera. Neither component I nor component II alone induced the fluid accumulation in intestinal loops. The intestinal loop activity was demonstrated with the culture supernatants of strains of Clostridium botulinum types C and D that produced C2 toxin, but not with culture supernatants of strains that did not. None of the botulinum type A through F neurotoxins induced fluid accumulation in mouse intestinal loops. The results indicate that, in addition to lethal and vascular permeability activities, C2 toxin has an enterotoxic activity for which the cooperation of components I and II is necessary. The fluid accumulation in intestinal loops inoculated with C2 toxin was not diminished by removal of the toxin from the loops. Moreover, the secretory response was positive when intestinal lumina were exposed to component II followed by the removal of the component and inoculation with component I, but it was negative when the intestinal lumina were exposed to component I followed by the removal of the component and inoculation with component II. These results suggest that the secretory response of mouse intestinal loops to C2 toxin is induced by the binding of component II to the epithelial cell surfaces of the intestines and the consequent binding or penetration of component I into the cells.

Lethal and vascular permeability activities of botulinum C2 toxin induced by separate injections of the two toxin components.

Two components, designated I and II, of botulinum C2 toxin were injected separately into the same animal. The intravenous injection of one component at different time intervals after intravenous injection of the other component, irrespective of the sequence, was lethal to mice. When components I and II were injected intradermally into separate sites, vascular permeability increased only at the site where component II was injected. The sizes of blued areas were smaller with increased distance between the injection sites of components I and II. When one component was injected intravenously and the other intradermally, an increase in vascular permeability was induced at the intradermal site of injection of component II but not at that of component I. These results indicate that the simultaneous injection of components I and II is not always required to elicit the biological activity of C2 toxin. The vascular permeability response induced by separate injections of the two toxin components suggests that the activity of C2 toxin results from component II binding to the tissue around its injection site and component I recognizing the altered tissue.

Oral toxicities of Clostridium botulinum type A and B toxins from different strains.

The production and the oral toxicity for mice of Clostridium botulinum type A and B toxins of different strains were studied. All five type B strains produced both 16S (large or L) and 12S (medium or M) toxins, although the relative amounts varied with the strains. The culture supernatant of type B Okra strain was the most potent in oral toxicity. The L toxin of this culture was about 700 times more toxic in feeding tests with mice than the L toxin from type B strain NH-2, whereas the M toxins of the two strains had the same oral toxicity. These results indicate that the oral toxicity of type B toxin varies with the culture strain. Oral toxicities of L toxin produced by type A strains 62A and 97 were comparable but were 10 times higher than those of their M toxins. Hybrids of toxic and nontoxic components separated from L toxins of type B strains Okra and NH-2 revealed that the high oral toxicity of the B-L toxin of strain Okra is attributable not to the toxic but to the nontoxic component of the toxin. The present study suggests that the 16S molecular-sized toxin elaborated by a certain strain of C. botulinum type B is implicated in the high fatality rate in type B human botulism.

Binding of the two components of C2 toxin to epithelial cells and brush borders of mouse intestine.

C2 toxin elaborated by Clostridium botulinum types C and D is composed of two nonlinked protein components and has enterotoxic activity, for which the cooperation of these two components is necessary. In the present study, the binding of components I and II, the two components of C2 toxin, to isolated epithelial cells and brush borders of mouse intestine was examined. Immunofluorescence studies showed that component II, either trypsinized (T-II) or untrypsinized (UT-II), bound to the cells and the brush borders of mouse intestine, whereas component I alone did not. The binding of I was observed only when the cells and the brush borders were reacted with T-II, but not when they were reacted with UT-II. These results are consistent with the fact that the biological activities of C2 toxin are elicited by the combination of I and T-II, but not of I and UT-II. The in vitro binding of I and II to isolated brush borders of mouse intestinal cells also showed similar binding characteristics. The binding of I and II to brush borders was rapid and not temperature dependent. Ultracentrifugal analysis revealed that both I and T-II bound to microvillous membranes of the intestinal cells. The data from the present study indicate that the enterotoxic activity of C2 toxin is initiated by the binding of T-II to the microvillous membrane of intestinal cells followed by that of I, for which the site of the cell membrane is induced by the binding of T-II, but not of UT-II.