Molecular characterization of the clusters of genes encoding the botulinum neurotoxin complex in clostridium botulinum (Clostridium argentinense) type G and nonproteolytic Clostridium botulinum type B

The cluster of genes encoding components of the progenitor botulinum neurotoxin complex has been mapped and cloned in Clostridium botulinum type G strain ATCC 27322. Determination of the nucleotide sequence of the region has revealed open reading frames encoding nontoxic components of the complex, upstream of the gene encoding BoNT/G (botG). The arrangement of these genes differs from that in strains of other antigenic toxin types. Immediately upstream of botG lies a gene encoding a protein of 1198 amino acids, which shows homology with the nontoxic-nonhemagglutinin (NTNH) component of the progenitor complex. Further upstream there are genes encoding proteins with homology to hemagglutinin components (HA-17, HA-70) and a putative positive regulator of gene expression (P-21). Sequence comparison has shown that BoNT/G has highest homology with BoNT/B. The sequence of the BoNT-cluster of genes in non-proteolytic C. botulinum type B strain Eklund 17B has been extended to include the complete NTNH and HA-17, and partial HA-70 gene sequences. Comparison of NTNH/G with other NTNHs reveals that it shows highest homology with NTNH/B consistent with the genealogical affinity shown between BoNT/G and BoNT/B genes.     

Botulinolysin, a thiol-activated hemolysin produced by Clostridium botulinum, inhibits endothelium-dependent relaxation of rat aortic ring

The effects of botulinolysin (Blyn), a thiol-activated hemolysin produced by Clostridium botulinum, on contractility of rat aortic ring were studied in order to clarify an underlying mechanism of vasoconstriction by the toxin observed previously as an increase in perfusion pressure in isolated rat organs. Blyn (30 hemolytic units/ml; HU/ml) itself did not elicit any apparent change in resting tension of the ring. Contractile tension elicited by a high concentration of phenylephrine in endothelium-intact rings increased significantly after treatment with Blyn (30 HU/ml), while phenylephrine-induced contraction of endothelium-denuded rings was not influenced by toxin treatment. In rings with intact endothelium, acetylcholine (ACh)-induced relaxation was significantly inhibited after treatment with Blyn (30, 10, 1 HU/ml). In contrast, relaxation of denuded rings by sodium nitroprusside was not affected by toxin treatment (30 HU/ml). Arginine (10(-4) M) partly reversed the inhibition of ACh-induced relaxation by the toxin (1 HU/ml). Endothelium-dependent relaxation by histamine or adenosine triphosphate was also inhibited by Blyn (1 HU/ml), but the relaxation elicited by calcium ionophore A23187 was not influenced by the toxin. The results indicate that Blyn acts on endothelium and inhibits agonist-induced endothelium-dependent relaxation of blood vessels.     

Inactivation of Clostridium botulinum toxin by ruminal microbes from cattle and sheep

Toxin from Clostridium botulinum type C was rapidly inactivated during incubation in vitro with ruminal contents from either a cow or a sheep. Fractions of ruminal contents from which cells had been removed by high-speed centrifugation did not inactivate toxin. Inactivation was associated with fractions containing bacteria, whereas fractions containing protozoa and relatively few bacteria were much less active. This activity may help explain the relatively greater tolerance by ruminants to oral doses of botulinum toxin than to toxin administered by other routes. The results are also pertinent to assays for botulinum toxin from gastrointestinal samples obtained postmortem.     

Mosaic type of the nontoxic-nonhemaggulutinin component gene in Clostridium botulinum type A strain isolated from infant botulism in Japan

The gene encoding the nontoxic-nonhemaggulutinin (NTNH) component was amplified by the PCR technique using two primer sets and the DNA template from Clostridium botulinum type A strain 7I03-H isolated from infant botulism in Japan. The nucleotide sequence revealed that the NTNH gene was composed of 1,193 amino acids with a molecular weight of 130868.08. Furthermore, the N-terminal half side and C-terminal half side of the NTNH component were similar to the NTNH component of type C and type A, respectively. These results indicate that the NTNH component gene codes the mosaic NTNH component composed of type A and type C. The hemaggulutinin gene, aha, and ORF-22 gene, orf-22a, were undetectable in the region upstream of the NTNH component gene, ant. Therefore, orf-22a is not thought to play a key role in the expression of botulinum type A progenitor toxin gene.     

Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis

The actin ADP-ribosylating Clostridium botulinum C2 toxin is a binary toxin composed of the binding component C2II and the enzyme component C2I. C2I ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we studied the structure-function relationship of C2I by site-directed mutagenesis. Exchange of Glu389 to glutamine caused the complete loss of ADP-ribosyltransferase and NAD-glycohydrolase activities of C2I. In contrast, exchange of Glu387 to glutamine blocked ADP-ribosyltransferase but not NAD-glycohydrolase activity. Whereas photoaffinity labeling of the double mutant E387Q/E389Q C2I with [carbonyl-14C]NAD was blocked, labeling of the single C2I mutants was reduced (E389Q) or not changed (E387Q). Exchange of the STS motif (amino acid residues 348-350) of C2I caused a decrease in transferase activity by more than 99 (S348A) and 90% (T349V), or did not affect activity (S350A). Exchange of Arg299 and Arg300 to lysine reduced transferase activity to <0.1 and approximately 35% of wild-type activity. The data indicate that the amino acid residues Glu389, Glu387, Ser348, and Arg299, which are conserved in various prokaryotic and eukaryotic arginine-modifying ADP-ribosyltransferases, are essential for ADP-ribosyltransferase activity of the enzyme component of C. botulinum C2 toxin.     

Analysis of the botulinum neurotoxin type F gene clusters in proteolytic and nonproteolytic Clostridium botulinum and Clostridium barati

The purpose of this in vitro study was to compare both apical and coronal dye penetration when Ketac-Endo and AH-26 sealers were used with laterally condensed gutta percha. Crowns were removed from 28 teeth and the root canals were biomechanically prepared. The teeth were divided into two groups of 12-teeth each and a control group of 4 teeth. Root canals in the two experimental groups were filled with laterally condensed gutta percha and either Ketac-Endo or AH-26 sealer. The Ketac-Endo group had the coronal 3 mm of gutta percha and sealer removed and the resultant cavity was filled with Ketac-Endo alone. After the sealers had set, the root surfaces were coated with nail varnish except at the apex and at the coronal end. Positive controls had no root fillings and were coated with nail varnish in the same manner while the negative controls were sealed apically and coronally with Cavit prior to sealing the entire external root surface with nail varnish. Specimens were placed in 2% methylene blue dye in a vacuum of 660 mm of mercury for five minutes and then left immersed for a further two days. The roots were vertically sectioned to determine the following mean levels of dye penetration: Ketac-Endo, 1.08 mm apically and 6.29 mm coronally; AH-26, 0.75 mm apically and 6.67 mm coronally. Positive controls had total leakage and negative controls had no leakage. This study demonstrated that the apical and coronal seals obtained with Ketac-Endo and AH-26 were not significantly different although the apical seal obtained with each material was significantly better than the corresponding coronal seal.     

The inhibition of Clostridium botulinum type C by other bacteria in wetland sediments

Bacteria with inhibitory activity against Clostridium botulinum type C were isolated from 32% of sediment samples (n = 1600) collected from 10 marshes in a northern California wetland over a 12 mo period. Aerobic and anaerobic bacteria with inhibitory activity were isolated from 12% and 23% of the samples, respectively. Bacteria with inhibitory activity were isolated from all 10 study sites and throughout the year. This study demonstrates that bacteria with inhibitory activity against C. botulinum type C occur naturally in wetland sediments.     

Treatment of recurrent dislocation of the temporomandibular joint with type A botulinum toxin

A case is reported of a 56-year-old woman who suffered from recurrent dislocations of the temporomandibular joint (TMJ) secondary to an exacerbated tetraspastic syndrome of multiple sclerosis. Following chemical denervation of the masseter and pterygoid muscles with injections of type A botulinum toxin, no further dislocations occurred for periods of up to four months. The treatment has been repeated five times. Some of the indications and possible adverse reactions to this therapy are discussed and comparisons made with other, conventional methods for managing recurrent dislocation of the TMJ.     

Investigation of the ability of proteolytic Clostridium botulinum to multiply and produce toxin in fresh Italian pasta

OBJECTIVE: To assess the outcomes of anesthesia and surgery for men and women 100 years of age and older. DESIGN: Retrospective cohort study in the 20-year time period from 1975 to 1994, with follow-up through 1995. SETTING: Mayo-affiliated hospitals and Olmsted Community Hospital, Rochester, Minnesota. PARTICIPANTS: All men and women 100 years of age and older who underwent surgery at a participating hospital. MEASUREMENTS: Forty-eight-hour and 30-day perioperative morbidity and mortality; long-term survival. RESULTS: Thirty-one men and women aged 100 to 107 years underwent 42 procedures. One major complication (3%) within 48 hours was observed. The 48-hour, 30-day, and 1-year mortality rates were 0%, 16.1%, and 35.5%, respectively. When compared with survival rates for age-, gender-, and calendar year of birth-matched peers from the general population, the survival rate for centenarians who underwent surgery and anesthesia was comparable to the rate expected. CONCLUSION: These data suggest that people 100 years of age and older who have operable diseases or injuries should not be denied surgical interventions because of perceived risks associated with their advanced age.     

Xanthofulvin, a novel semaphorin inhibitor produced by a strain of Penicillium

A new semaphorin inhibitor xanthofulvin was isolated from the cultured broth of a fungus Penicillium sp. SPF-3059 along with a known compound vinaxanthone by solvent extraction and bioassay-guided fractionation. The tautomeric structure of xanthofulvin was determined by spectroscopic analyses. The two compounds exhibited significant semaphorin inhibitory activity with IC50 values of 0.09 and 0.1 microg/ml, respectively, in semaphorin3A-induced growth cone collapse assay using cultured chick dorsal root ganglia neurons.     

Microbiological quality and the inability of proteolytic Clostridium botulinum to produce toxin in film-packaged fresh-cut cabbage and lettuce

The production of toxin by a 10-strain mixture of proteolytic Clostridium botulinum in fresh produce packaged in polyethylene films having high (7,000 cc/m2/24 h; HOTR) and low (3,000 cc/m2/24 h; LOTR) relative oxygen permeability was determined. Shredded cabbage and lettuce inoculated with approximately 10(2) spores/g were placed in bags composed of the two films (1.4 kg/bag), and the bags were then vacuum sealed. Produce was stored at 4, 13, and 21 degrees C for up to 21 (cabbage) or 28 (lettuce) days and analyzed periodically. At each sampling time, the gas composition within the bags, pH of the produce, and microbial populations (total aerobic and anaerobic microorganisms, lactic acid bacteria, psychrotrophic bacteria, and yeasts and molds) were determined. In addition, the presence of botulinal toxin was determined using the standard U.S. Food and Drug Administration mouse bioassay protocol. Bags made of HOTR film prolonged sensory quality of cabbage and lettuce, especially at 13 and 4 degrees C. Packaging material had an effect on the growth of various groups of microorganisms; however, there was not a general trend. For example, lettuce packaged in HOTR bags had higher aerobic microbial populations than that packed in LOTR, but no significant difference (P < or = 0.05) was observed with cabbage. Growth of psychrotrophic bacteria was greater in vegetables packaged in HOTR film while growth of yeasts and molds was not affected by either packaging film. Most differences in microbial populations in produce packaged in LOTR and HOTR films were less than 1 log10 CFU/g. Botulinal toxin was not detected in cabbage or lettuce packaged in either film or stored under any test condition.     

Gating and permeability of ion channels produced by botulinum toxin types A and E in PC12 cell membranes

Botulinum neurotoxin (BoNT) is known to produce cationic channels in artificial bilayers. This study examined ion channels formed by BoNT in native membranes from cultured PC12 cells under conditions approximating those thought to occur during toxin internalization. Membrane patches were excised from PC12 cells using patch electrodes and exposed to symmetrical solutions containing either 200 mM CsCl, RbCl or KCl. The patch pipettes also contained 1-5 microg/ml BoNT buffered to pH 5.3 while the bath solutions were buffered to pH 7.0. In the presence of toxin, bursts of ion channel openings were observed. These toxin-induced channels were most active with a negative voltage applied to the same side as the toxin (cis). The increased activity at negative voltages was due to an increase in mean open time of e-fold per 120 mV and a decrease in mean closed time between bursts of e-fold per 110 mV. The shorter mean closed time within a burst was independent of membrane voltage. While BoNT-induced ion channels started as a single conductance level of 27 pS (KCl), 34 pS (RbCl) or 46 pS (CsCl) they typically increased in roughly equal steps to five or more times the original channel conductance. These higher conductance BoNT 'channels' opened and closed synchronously and could be distinguished from superposition of multiple independent channels. Despite differences in putative transmembrane sequences between BoNT/A and BoNT/E, both serotypes evidenced the same channel conductance and mean open time.     

Has botulinum toxin type A a place in the treatment of spasticity in spinal cord injury patients?

OBJECTIVE: To present and discuss treatment of severe spasms related to spinal cord injury with botulinum toxin type A. DESIGN: A 2-year follow-up study of an incomplete T12 paraplegic patient, who was reluctant to undergo intrathecal baclofen therapy, presenting severe painful spasms in his lower limbs treated with intramuscular injections of botulinum toxin type A. SETTING: Department of Physical Medicine and Rehabilitation, H?pital de Gravelone, Sion, Switzerland. SUBJECT: Single patient case report. MAIN OUTCOME MEASURE: Spasticity, spasms and pain measured with the modified Ashworth scale, spasm frequency score and visual analogue scale. RESULTS: Treatment of spasticity with selective intramuscular injections of botulinum toxin type A resulted in subjective and objective improvement. CONCLUSION: Botulinum toxin type A has its place in the treatment of spasticity in spinal cord injury patients. This treatment is expensive and its effect is reversible. It can complement intrathecal baclofen in treating upper limb spasticity in tetraplegic patients. Tolerance does occur to the toxin. Although high doses of the product are well tolerated, the quantity should be tailored to the patient's need. The minimal amount necessary to reach clinical effects should be adhered to and booster doses at short period intervals should be avoided.     

HP17, a new pigment-like antibiotic produced by a new strain of Spirillospora

Spirillospora strain 719 produces several antibiotics. On solid and liquid media, a deep red pigments is formed and diffuses throughout the culture. It was extracted with methanol from the mycelium cake and from the fermentation broth after precipitation at pH 2 and purified using TLC and HPLC. Its u.v. absorption spectrum and its physicochemical characteristics place this antibiotic in the 3.3.2.2.8 of the Berdy et al. classification. In most respects, it resembles proteinaceous pigment from Spirillospora 1655 and 1309-b that was studied and named spirillomycin. However, HP17 differs from spirillomycin principally in molecular weight and chemical nature.     

AH17, a new non-polyenic antifungal antibiotic produced by a strain of Spirillospora

An antibiotic (AH17) was produced by Spirillospora strain 719. This substance was obtained only from the broth filtrate after precipitation with acetic acid followed by extraction with n-butanol. Its purification was carried out by thin layer chromatography on silica gel followed by HPLC procedures. It showed activity against Gram-positive and Gram-negative bacteria and fungi. The spectroscopic and chemical properties were examined and indicated that AH17 was a quinone. It seems that this is a new antibiotic from Spirillospora.     

Conservative prediction of time to Clostridium botulinum toxin formation for use with time-temperature indicators to ensure the safety of foods

Integrating-type time-temperature indicators (TTIs) may be utilized to warn food processors and consumers about storage conditions that may have rendered a food potentially hazardous. As an example of how integrated TTIs could be manufactured to emulate an infinite set of time-temperature situations, a set of conditions which have supported C. botulinum growth and toxin production was compiled. The time-temperature curve representing conservative times required for toxin formation was constructed with data from literature relating to toxin formation as a function of temperature in any media or food product. This set of critical time-temperature data is fit by a conservative empirical relationship that can be used to predict combinations of incubation times and storage temperatures that represent a potential health risk from C. botulinum in foods. A TTI could be constructed to indicate deviation from such a given set of conditions to bring attention to foods that may have been exposed to potentially hazardous temperatures with respect to C. botulinum toxin formation.     

Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins

Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.