Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagesesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2

An important characteristic of the human group IIA secreted phospholipase A(2) (IIA PLA(2)) is the extremely low activity of this enzyme with phosphatidylcholine (PC) vesicles, mammalian cell membranes, and serum lipoproteins. This characteristic is reflected in the lack of ability of this enzyme to bind productively to zwitterionic interfaces. Part of the molecular basis for this lack of activity is an absence of tryptophan, a residue with a known preference for residing in the interfacial region of zwitterionic phospholipid bilayers. In this paper we have replaced the eight residues that make up the hydrophobic collar on the interfacial binding surface of the enzyme with tryptophan. The catalytic and interfacial binding properties of these mutants have been investigated, particularly those properties associated with binding to and hydrolysis of zwitterionic interfaces. Only the insertion of a tryptophan at position 3 or 31 produces mutants that significantly enhance the activity of the human IIA enzyme against zwitterionic interfaces and intact cell membranes. Importantly, the ability of the enzyme mutants to hydrolyze PC- rich interfaces such as the outer plasma membrane of mammalian cells was paralleled by enhanced interfacial binding to zwitterionic interfaces. The corresponding double tryptophan mutant (V3,31W) displays a specific activity on PC vesicles comparable to that of the human group V sPLA2. This enhanced activity includes the ability to interact with human embryonic kidney HEK293 cells, previously reported for the group V enzyme [Kim, Y. J., Kim, K. P., Rhee, H. J., Das, S., Rafter, J. D., Oh, Y. S., and Cho, W. (2002) J. Biol. Chem. 277, 9358-9365

The antibacterial properties of secreted phospholipases A(2) - A major physiological role for the group IIA enzyme that depends on the very high pI of the enzyme to allow penetration of the bacterial cell wall

The antibacterial properties of human group IIA secreted phospholipase A(2) against Gram-positive bacteria as a result of membrane hydrolysis have been reported. Using Micrococcus luteus as a model system, we demonstrate the very high specificity of this human enzyme for such hydrolysis compared with the group IB, IIE, lIF, V, and X human secreted phospholipase A(2)s. A unique feature of the group IIA enzyme is its very high pI due to a large excess of cationic residues on the enzyme surface. The importance of this global positive charge in bacterial cell membrane hydrolysis and bacterial killing has been examined using charge reversal mutagenesis. The global positive charge on the enzyme surface allows penetration through the bacterial cell wall, thus allowing access of this enzyme to the cell membrane. Reduced bacterial killing was associated with the loss of positive charge and reduced cell membrane hydrolysis. All mutants were highly effective in hydrolyzing the bacterial membrane of cells in which the cell wall was permeabilized with lysozyme. These same overall characteristics were also seen with suspensions of Staphylococcus aureus and Listeria innocua, where cell membrane hydrolysis and antibacterial activity of human group IIA enzyme was also lost as a result of charge reversal mutagenesis

Effect of absorbable membranes on sandwich bone augmentation

Objectives : This study was conducted to evaluate the effect of barrier membranes on sandwich bone augmentation (SBA) for the treatment of implant dehiscence defects. Material and methods : Twenty-six implant-associated buccal dehiscence defects in 22 patients were treated according to the SBA concept – mineralized human cancellous allograft (inner layer), mineralized human cortical allograft (outer layer) and coverage with barrier membrane. The defects were randomly assigned to the bovine collagen membrane (BME) group; acellular dermal matrix (ADM) group; and no membrane group. Measurements at baseline and 6 months re-entry included defect height (DH: from smooth–rough junction to the most apical part of the defect), defect width (DW: at the widest part of the defect), and horizontal defect depth (HDD: at three locations – smooth–rough junction, middle, and most apical portion of the defect). All measurements were taken from a reference stent. Statistical analyses were performed for comparison of intra- and inter-group comparisons. Results : All implants placed were successfully osseointegrated. DH at baseline for three groups were not significantly different ( P =0.858). Mean % DH reductions for ADM, BME, and control groups at 6 months were 73.9±17.6%, 68.1±30.1%, and 63.6±23.9%, respectively, with no significant difference among the groups ( P =0.686). Mean horizontal bone gain, however, was significantly greater for membrane groups (1.7 mm for ADM, 1.6 mm for BME) compared with control group (1 mm) ( P =0.044). Implant exposure resulted in significant reduction in total height gain (79.1±14.3% vs. 57±23.5%, P =0.021). Conclusions : Within the limit of this study, it is concluded that SBA technique achieved predictable clinical outcomes. The addition of absorbable membranes enhanced bone gain in thickness compared with membrane-treated sites. To cite this article: Park S-H, Lee K-w, Oh T-J, Misch CE, Shotwell J, Wang H-L. Effect of absorbable membranes on sandwich bone augmentation. Clin. Oral Impl. Res . 19 , 2008; 32–41. doi: 10.1111/j.1600-0501.2007.01408.xPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74133/1/j.1600-0501.2007.01408.x.pd