Background: Imatinib mesylate is a small molecule targeted at dysregulated protein-tyrosine kinase. Mutation of c-kit exon 11, which induces constitutive phosphorylation of KIT, is one of the mechanisms for the development or progression of mast cell tumor (MCT) in dogs. The purpose of this study was to examine the therapeutic potential of imatinib mesylate in canine MCT.Hypothesis: Imatinib mesylate has activity against MCT in dogs, and response to treatment can be correlated to presence of mutation within exon 11 of c-kit.Animals: Twenty-one dogs with MCT with gross tumor burden and median tumor size of 7.2 cm (range, 1.0-25.3 cm) before treatment.Methods: Tumors were analyzed for mutation of c-kit exon 11. Imatinib mesylate was administered PO to the dogs at a dose of 10 mg/kg daily for 1-9 weeks.Results: Ten of 21 dogs (48%) had some beneficial response to imatinib mesylate treatment within 14 days of treatment initiation. All 5 dogs with a demonstrable c-kit mutation in exon 11 responded to the drug (1 complete remission, 4 partial remission).Conclusions and Clinical Importance: Imatinib mesylate has clinical activity against MCT in dogs. Response could not be predicted based on presence of absence of a mutation in exon 11 of c-kit.

Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin to phosphocholine and ceramide in a divalent metal ion-dependent manner. Bc-SMase is a homologue of mammalian neutral SMase (nSMase) and mimics the actions of the endogenous mammalian nSMase in causing differentiation, development, aging, and apoptosis. Thus Bc-SMase may be a good model for the poorly characterized mammalian nSMase. The 2؉ or Co 2؉ may provide a common structural framework applicable to phosphohydrolases belonging to the DNase I-like folding superfamily. In addition, the structural features and site-directed mutagenesis suggest that the specific ␤-hairpin with the aromatic amino acid residues participates in binding to the membrane-bound sphingomyelin substrate.Sphingomyelinase (SMase, 4 EC 3.1.4.12) from Bacillus cereus (BcSMase) exhibits both phospholipase C activity, hydrolyzing sphingomyelin (SM) to phosphocholine and ceramide, and hemolytic activity. Both Bc-SMase activities occur in a divalent metal ion-dependent manner (1-3). Bc-SMase requires Mg 2ϩ for sphingomyelin hydrolytic activity and an additional Ca 2ϩ for hemolytic activity (1-6). Mutagenesis studies have shown that Glu-53 is the essential Mg 2ϩ -binding amino acid (5).The catalytic mechanism of the sphingomyelin hydrolytic activity remains to be elucidated in atomic detail, as there are no crystal structures of SMase in complex with the essential divalent metal ions. The sphingomyelin hydrolytic activity of Bc-SMase is believed to proceed in the manner of acid base catalysis, in which His-296 is proposed to generate an activated water and the essential Mg 2ϩ ion at Glu-53 is suggested to stabilize a negatively charged transition state. The proposed catalytic mechanism of Bc-SMase is similar to that of bovine DNase I. In fact, Bc-SMase and bovine DNase I are homologous proteins and share a common architecture of conserved putative catalytic amino acid residues (7). However, the proposed catalytic mechanism does not fully explain the role of the essential divalent metal ion, i.e. the divalent metal ion type dependence for hydrolytic catalysis, because of the lack of the bound essential metal ions in all the currently available structures.Bc-SMase and neutral sphingomyelinase (nSMase) in mammalian cells share similar metal ion dependence and considerable amino acid sequence identity (20%), including conserved residues involved in divalent metal ion binding, and are thus believed to have a similar hydrolytic mechanism. SMases in mammalian cells are classified into the following three groups according to the optimum pH of the SM hydrolytic activity: neutral SMase, acid SMase, and alkaline SMase (8). The detailed mechanism of the sphingomyelin hydrolysis activity of Bc-SMase may provide insight into sphingolipid metabolism in mammalian cells (9 -11).The only available structure of SMase is from the bacterium, Listeria ivanovii, reported recently (7). The bacterial SMase was confirmed to be a member of the DNase I-like folding superfamily (12)(13)(14)...

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Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins.

Bacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H2O2 and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.