Cryo-electron microscopy of two-dimensional crystals of reduced type B botulinum neurotoxin

1992 ◽  
Vol 50 (1) ◽  
pp. 530-531
Author(s):  
P. F. Flicker ◽  
V.S. Kulkarni ◽  
J. P. Robinson ◽  
G. Stubbs ◽  
B. R. DasGupta
Keyword(s):  
Disulfide Bonds ◽  
Clostridium Botulinum ◽  
Structural Changes ◽  
Two Dimensional ◽  
Type B ◽  
Single Chain ◽  
Muscle Paralysis ◽  
Cryo Electron Microscopy ◽  
Disulfide Linkages ◽  
Intracellular Action

Botulinum toxin is a potent neurotoxin produced by Clostridium botulinum. The toxin inhibits release of neurotransmitter, causing muscle paralysis. There are several serotypes, A to G, all of molecular weight about 150,000. The protein exists as a single chain or or as two chains, with two disulfide linkages. In a recent investigation on intracellular action of neurotoxins it was reported that type B neurotoxin can inhibit the release of Ca++-activated [3H] norepinephrine only if the disulfide bonds are reduced. In order to investigate possible structural changes in the toxin upon reduction of the disulfide bonds, we have prepared two-dimensional crystals of reduced type B neurotoxin. These two-dimensional crystals will be compared with those of the native (unreduced) type B toxin.

Two dimensional crystals of botulinum toxin, serotype B

1989 ◽  
Vol 47 ◽  
pp. 1034-1035
Author(s):  
D.G. Morgan ◽  
B.R. DasGupta ◽  
G. Stubbs ◽  
J.P. Robinson
Keyword(s):  
Electron Microscope ◽  
Botulinum Toxin ◽  
Clostridium Botulinum ◽  
Binding Property ◽  
Two Dimensional ◽  
Type B ◽  
Acetyl Choline ◽  
Sodium Phosphate Buffer ◽  
Carbon Coated ◽  
Serotype B

Botulinum toxin is a powerful, protein neurotoxin produced by Clostridium botulinum which exerts its toxic action by inhibiting the release of acetyl choline. There are several immunologically distinguishable types of botulinum toxin and most of these have been shown to bind the ganglioside GTlb. The ganglioside binding property of these neurotoxins has allowed us to prepare two dimensional crystals of serotypes A, B, and E. We report here our preliminary observations of two dimensional crystals of serotype B.Type B botulinum toxin was purified by methods reported by DasGupta and Woody. The two dimensional crystals were prepared by slightly modified procedures used previously to prepare similar crystals of tetanus and cholera toxins. Purified toxin was dialyzed into citric acid-sodium phosphate buffer at pH 4.0 to 6.5 and ionic strength of about 0.04. Dialyzed toxin was placed into the wells of microtiter dishes in 20 μl volumes at a concentration of fifty to one hundred μgm per ml. The toxin solutions were then layered with one to two μl of a solution of one to two mg per ml of egg lecithin (Sigma Cat. No. P- 2772) in chloroform containing five to ten percent by weight of the ganglioside GT1B (Supelco Cat. No. 4-6035). The microtiter dishes were then placed in the cold and crystallization was allowed to proceed for one to four days at 4°C. The crystals were then picked up on carbon coated electron microscope grids, negatively stained with one to two percent uranium acetate and examined in the electron microscope.

scholarly journals Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes.

1994 ◽  
Vol 269 (14) ◽  
pp. 10498-10503 ◽  
Author(s):  
T. Nishiki ◽  
Y. Kamata ◽  
Y. Nemoto ◽  
A. Omori ◽  
T. Ito ◽  
...  
Keyword(s):  
Rat Brain ◽  
Clostridium Botulinum ◽  
Type B ◽  
Rat Brain Synaptosomes ◽  
Protein Receptor ◽  
Botulinum Type ◽  
Brain Synaptosomes

scholarly journals Development of a Combined Selection and Enrichment PCR Procedure for Clostridium botulinum Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs

2001 ◽  
Vol 67 (10) ◽  
pp. 4781-4788 ◽  
Author(s):  
Maria Dahlenborg ◽  
Elisabeth Borch ◽  
Peter Rådström
Keyword(s):  
Sample Preparation ◽  
Fecal Sample ◽  
Clostridium Botulinum ◽  
Geographical Location ◽  
Type B ◽  
Fecal Samples ◽  
Pcr Assays ◽  
Combined Selection ◽  
Spore Load ◽  
Slaughtered Pigs

ABSTRACT A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymeraserTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 103 spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.

scholarly journals Type B trichothecenes-the relationship between slight structural changes and toxicity

2016 ◽  
Vol 66 (1) ◽  
pp. 45-55
Author(s):  
Tadahiro Suzuki ◽  
Yumiko Iwahashi
Keyword(s):  
Structural Changes ◽  
Type B ◽  
The Relationship

Human platelets contain forms of factor V in disulfide-linkage with multimerin

2004 ◽  
Vol 92 (12) ◽  
pp. 1349-1357 ◽  
Author(s):  
Nola Fuller ◽  
Shilun Zheng ◽  
Frédéric Adam ◽  
Samira Jeimy ◽  
Ian Horsewood ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Human Platelets ◽  
Factor V ◽  
Single Chain ◽  
Platelet Factor ◽  
Disulfide Linkage ◽  
Factor Va ◽  
Plasma Factor ◽  
Large Platelet ◽  
Large Factor

SummaryFactor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factorV is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage.The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

Potential for Growth of Nonproteolytic Types of Clostridium botulinum in Pasteurized Restructured Meat Products: A Review1

1985 ◽  
Vol 48 (3) ◽  
pp. 265-276 ◽  
Author(s):  
J. SIMUNOVIC ◽  
J.L. OBLINGER ◽  
J.P. ADAMS
Keyword(s):  
Heat Resistance ◽  
Causative Agent ◽  
Clostridium Botulinum ◽  
Meat Products ◽  
Type B ◽  
The U.S ◽  
Restructured Meat

Type E and nonproteolytic type B strains of Clostridium botulinum can grow and produce toxin at temperatures below 5°C. Recent publications describing the greater heat resistance of nonproteolytic type B C. botulinum spores than type E spores are discussed in relation to suitable proess lethalities required for a safe pasteurized product. The incidences of botulism in Europe caused by nonproteolytic type B spores were compared to the lack of such incidences in the U.S. and to published procedures for isolating the causative agent for botulism. The incidence of C. botulinum spores in meat products in the U.S. also is reviewed.

scholarly journals Study on potential Clostridium botulinum growth and toxin production in Parma ham

2016 ◽  
Vol 5 (2) ◽  
Author(s):  
Giuseppe Merialdi ◽  
Mattia Ramini ◽  
Giovanni Parolari ◽  
Silvana Barbuti ◽  
Maria Angela Frustoli ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Toxin Production ◽  
Limiting Factor ◽  
In Vitro Test ◽  
Type B ◽  
Potential Growth ◽  
Parma Ham ◽  
Nacl Concentration ◽  
Vitro Test

The objective of this study was to investigate <em>Clostridium botulinum</em> growth and toxin production in the industrially manufactured Italian Parma ham. The study focuses on the Parma ham production phase identified as maximum risk to <em>C. botulinum</em> proliferation, <em>i.e.</em> the transition from cold phase (salting and resting) to a phase carried out at temperature between 15 and 23°C (drying). A preliminary in vitro test was carried out in order to verify the capability of 6 <em>C. botulinum</em> strains (1 type A, 4 type B, and 1 type E strains) to grow in conditions of temperature, pH and NaCl concentration comparable to those of the beginning stage of ham drying. Five <em>C. botulinum</em> strains grew at 20°C and pH 6, four strains produced toxin when inoculated at a concentration equal to 10<sup>3</sup> cfu/mL at NaCl concentration of 4%, while when the inoculum concentration was 10 cfu/mL, NaCl concentration of 3% resulted the toxin-genesis limiting factor. An experimental contamination with a mixture of the 5 <em>C. botulinum</em> strains selected by the preliminary in vitro test was performed on 9 thighs inoculated at the end of the resting phase. The study was designed to evaluate the potential growth and toxin production in extremely favourable conditions for the bacterium. Type B proteolytic <em>C. botulinum</em> toxin was produced after 14 days of incubation at 20°C in 2 thighs characterised by high weight, low number of days of resting and anomalous physiochemical characteristics [one for very low NaCl concentration (1.59%), the other for elevated pH (6.27) and both for high water activity values (&gt;0.970)]. The results of this research confirm that the cold resting step is a critical phase in the production process of Parma ham for the investigated hazard. Based on the present study, the long resting phase adopted in the manufacturing of Parma ham is proven effective to prevent the growth of <em>C. botulinum</em>, an event which could not otherwise be excluded if the hams were processed under less stringent technological conditions.

Differentiation between Types and Strains of Clostridium botulinum by Riboprinting

2000 ◽  
Vol 63 (10) ◽  
pp. 1347-1352 ◽  
Author(s):  
GUY E. SKINNER ◽  
STEVEN M. GENDEL ◽  
GEOFFREY A. FINGERHUT ◽  
HAIM A. SOLOMON ◽  
JODIE ULASZEK
Keyword(s):  
Clostridium Botulinum ◽  
Hazard Analysis ◽  
Control Point ◽  
Egg Yolk ◽  
Type B ◽  
Critical Control Point ◽  
Microbial Characterization ◽  
Automated Ribotyping ◽  
Degree Of Differentiation ◽  
Foodborne Botulism

The ability of automated ribotyping to differentiate between major types and individual strains of Clostridium botulinum was tested using the Qualicon Riboprinter Microbial Characterization System. Pure spores of C. botulinum type A, proteolytic type B, nonproteolytic type B, and type E strains were inoculated onto modified anaerobic egg yolk agar and incubated 24 h at 35°C. Plates were rinsed with buffer (2 mM Tris + 20 mM EDTA) to remove vegetative cells that were heated for 10 min at 80°C, treated with a lysing agent, and ribotyped in the Qualicon Riboprinter utilizing the enzyme EcoRI. Riboprint patterns were obtained for 30 strains of the four major types of C. botulinum most commonly involved in human foodborne botulism. Proteolytic strains yielded the best and most consistent results. Fifteen ribogroups were identified among the 31 strains tested. Interestingly, in two cases, a single ribogroup contained patterns from isolates belonging to evolutionarily distinct Clostridium lineages. This degree of differentiation between strains of C. botulinum may be useful in hazard analysis and identification, hazard analysis and critical control point monitoring and validation, environmental monitoring, and in inoculation studies.

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