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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)...

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.

First generation, photo-crosslinkable dendrimers consisting of natural metabolites (i.e. succinic acid, glycerol, and β-alanine) and non-immunogenic poly(ethylene glycol) (PEG) were synthesized divergently in high yields using ester and carbamate forming reactions. Aqueous solutions of these dendrimers were photo-crosslinked with an eosin-based photo-initiator to afford hydrogels. The hydrogels displayed a range of mechanical properties based on their structure, generation size, and concentration in solution. All of the hydrogels showed minimal swelling characteristics. The dendrimer solutions were then photo-crosslinked in situ in an ex vivo rabbit osteochondral defect (3 mm diameter and 10 mm depth), and the resulting hydrogels were subjected to physiologically relevant dynamic loads. Magnetic resonance imaging (MRI) showed the hydrogels to be fixated in the defect site after the repetitive loading regimen. The ([G1]-PGLBA-MA) 2 -PEG hydrogel was chosen for the 6 month pilot in vivo rabbit study because this hydrogel scaffold could be prepared at low polymer weight (10wt%) and possessed the largest compressive modulus of the 10% formulations, a low swelling ratio, and contained carbamate linkages which are more hydrolytically stable than the ester linkages. The hydrogel treated osteochondral defects showed good attachment in the defect site and histological analysis showed the presence of collagen II and glycosaminoglycans (GAGs) in the treated defects. By contrast, the contralateral unfilled defects showed poor healing and negligible GAG or collagen II production. Good mechanical properties, low swelling, good attachment to the defect site, and positive in vivo results illustrate the potential of these dendrimerbased hydrogels as scaffolds for osteochondral defect repair. KeywordsOsteochondral defects; Cartilage; Tissue Engineering; Scaffolds; Hydrogel; Dendrimer; Dendritic Polymer Osteoarthritis (OA) is the most common form of arthritis, and affects over 20 million people each year. 1 Early treatments include the use of anti-inflammatory drugs and physical therapy to relieve pain and maintain motion. When these methods are no longer effective, they are followed by cartilage grafting to repair discrete chondral lesions and ultimately total joint arthroplasty for advanced arthritis.2 , 3 In the initial stages of OA, proteoglycans are lost from the cartilage tissue followed by loss of the collagenous proteins.2 ,4 These events lead to formation of small discrete lesions in the cartilage tissue. With disease progression these lesions increase in size and depth, eventually reaching the subchondral bone. 5-7 Next, bone marrow cells migrate to the defect site, affording a healing response consisting of the formation of fibrocartilage.8 Fibrocartilage is mechanically inferior to the native hyaline cartilage, and is thus not as effective for load-bearing at this location. This natural healing response is the basis NIH Public Access for many treatment strategies and surgical techniques to treat discrete osteoc...