Purification of Toxin A and Toxin B

Purification of Clostridium difficile toxins A and B is performed using a modified procedure according to Eichel-Streiber et ai, 1987. 1. A dialysis bag containing 900 ml of 0.9 NaCI in a total volume of 41 brain heart infusion (BHI, Difco) is inoculated with 100 ml of an overnight BHI culture of C. difficile strain VPI 10463. 2. After incubation under microaerophilic conditions at 37 C for 72 h the culture is centrifuged for 20min at 8000 g in a Sorvall GSA rotor to obtain the culture...

Regulated Exocytosis

All mammalian cells display constitutive secretion, which is thought to be regulated at the stage of the budding of microvesicles from the trans-Golgi network (Burgess and Kelly, 1987). In addition, the specialized secretory cells exhibit regulated secretion. The docking of secretory vesicles on, and their fusion with, the plasma membrane are processes that appear to share many features with intracellular vesicle fusion (Scheller, 1995). The control over regulated exocytosis is exerted through...

Application of the Snare Hypothesis to Insulin Exocytosis

Several of the proteins which, it has been suggested, regulate neurotransmitter release have recently been identified in the (3-cell, and for a number of them a function in insulin exocytosis has been established. The ubiquitous fusion factor NSF is present in the insulin-secreting cell line HIT-T15. In addition, aSNAP, also present in these cells, appears to be one of the factors in cytosol required for Ca2+-triggered insulin exocytosis (Kiraly-Borri et at., 1996). The v-SNARE VAMP-2 was...

Clostridium botulinum C3 Exoenzyme and Studies on Rho Proteins

The Rho family of small GTP-binding proteins functions to regulate the assembly of distinct actin structures in cells Rho regulates stress fiber assembly, Rae regulates lamellipodia protrusion and Cdc42 stimulates protrusion of the plasma membrane to form filopodia Ridley and Hall, 1992 Ridley et al., 1992 Nobes and Hall, 1995 Kozma et al., 1995 . All three regulate attachment of cells to the extracellular matrix via adhesive integrin structures Nobes and Hall, 1995 . We and others have used...

Bioassay

This assay is based on injection of toxin into live mammals usually mice and is used to check the potency lethality of a given toxin preparation. It has also been applied to the study of substances that antagonize the effect of the toxin by interfering with the initial steps of poisoning Bakry etal., 1991 Simpson etal., 1990 . Normally, the LD50 or the time to death is measured Boroff and Fleck, 1966 and compared with non-treated control animals. It is also possible to inject small amounts of...

Clostridium botulinum ADPribosyltransferase C3

ADP-ribosyltransferase C3 is an exoenzyme produced by several strains of Clostridium botulinum. It was serendipitously detected during screening for high producer strains of C. botulinum C2 toxin Aktories et al., 1987 Aktories et a ., 1988b . The novel ADP-ribosyltransferase was termed C. botulinum ADP-ribosyltransferase C3, because it proved to be distinct from C. botulinum neurotoxin CI and the actin ADP-ribosylating C. botulinum C2 toxin. C3 ADP-ribosylates members of the Rho protein family...