No abstract

To check and clarify existing data on receptor-interacting residues in the human C5a anaphylatoxin, we tested mutant C5 a proteins obtained by site-directed mutagenesis of a recombinant human C5a (rhC5a) cDNA clone for structural and functional integrity. Amino acid positions in three different regions of the molecule were investigated: Arg74 at the C-terminus, Arg40 and Pro45 located in the core region, and Lysl4 and Lysl9, Lys20 in the N-terminus. Des-Arg74-rhC5a displayed only a residual 3 -4% functional activity in the myeloperoxidase-release assay from human granulocytes while retaining the three-dimensional solution structure of wild-type (wt) -rhC5 a as shown by circular dichroism (CD) spectroscopy. Des-Arg74-rhC5a was able to activate the human C5 a receptor transiently expressed in Xenopus oocytes, but was inactive in the heterologous guinea pig (gp) ileum-contraction assay. These results reveal profound differences between the guinea pig and human C5a-receptor ligand-binding characteristics. Exchange of the core residue Arg40 by a glycine did not significantly affect functional C5a activity, in contrast to a previous observation [Mollison, K. W., Mandecki, W., Zuiderweg, E. P., Fayer, L., Fey, T. A., Krause, R. A., Conway, R. G., Miller, L., Edalji, R. P., Shallcross, M. A., Lane, B., Fox, J. L., Greer, J. & Carter, G. W. (1989) Identification of receptor-interacting residues in the inflammatory complement protein C5 a by sitedirected mutagenesis, Proc. Nutl Acad. Sci. USA 86, 292-2961, nor did exchange of the conserved Pro45 residue by the C3a analogue glutamic acid, a mutation expected to alter the whole geometry of the loop connecting helix I11 -helix IV (including Arg40) of the C5a molecule. Thus, participation of this loop in receptor interaction appears unlikely. While exchange of the N-terminal Lysl4 residue by alanine did not significantly affect functional activity, a double replacement of Lysl9 and Lys20 by alanine residues reduced activity more than 30-fold. These results c o n f m Lysl9 a n d or Lys20 as a putative receptor-interacting site, although we could not obtain a CD spectrum of this important mutant due to poor expression.The human C5a anaphylatoxin is a small protein of 74 amino acids which mediates a variety of cellular activation and release processes. It is generated at sites of inflammation with concomitant complement-system activation by proteolytic cleavage from the a chain of the complement factor C5. The major target cells are the granulocyte and macrophage, leading to chemotactic attraction of neutrophils, induction of the oxidative burst and release of lysosomal enzymes, histamine and interleukins. All responses are mediated by interaction with the membrane-bound C5a receptor, which has recently been cloned (for review, see Kohl and Bitter-Suermann, 1993).Identification of receptor-interacting residues in the human C5 a molecule are essential for the rational development of receptor antagonists which are of considerable therapeutic

Previous studies have shown that various factors such as ionic composition or pH of the extraction medium may significantly influence leaching of components from restorative materials. Therefore, it was the aim of this investigation to determine the release of fluoride from a resin-modified glass-ionomer cement (GIC) following storage in various extraction media, including an esterase buffer. Specimens of the resin-modified GIC, Fuji II LC, were stored for 144 h in deionized water, acidic buffer (pH 4.2), neutral buffer (pH 7.0), and neutral buffer supplemented with porcine liver esterase. Fluoride release into the various media was measured every 48 h over a 6-day period. In addition, activity of porcine esterase in neutral buffer (artificial saliva) was measured for up to 144 h. The data were statistically evaluated by three-way ANOVA using the Student-Newman-Keuls test (P < 0.05). It was found that esterase activity in neutral artificial saliva decreased during the first 24 h to approximately 40% of the baseline value and then remained constant for up to 6 days. Fluoride release into the various storage media varied significantly (P < 0.05). The highest amounts of fluoride were released into deionized water (30.9 ppm +/- 1.1) and acidic buffer (26.9 ppm +/- 0.7) after 48 h. In addition, significantly more fluoride leached into esterase-containing neutral artificial saliva (6.9 ppm +/- 0.2) than into neutral buffer without enzyme (6.3 ppm +/- 0.2) after 96 h. Our data indicate that fluoride release from the resin-modified GIC investigated may be increased under acidic conditions and by hydrolysis in saliva.