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The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

High transmission of slow-light in a photonic crystal (PC) waveguide (WG) using a hetero group-velocity (Ht-V(g)) PC-WG was proposed and experimentally investigated. The Ht-V(g) WG, which comprises a low-group-velocity (L-V(g)) PC-WG section between two identical high-group- velocity (H-V(g)) PC-WGs, is designed to decrease the impedance mismatch of the L-V(g) PC-WG. The increase in transmittance of a propagating pulse was confirmed in the Ht-V(g) PC-WG even in the vicinity of the band-gap, whereas the homogeneous PC-WG showed a gradual decrease in transmittance with the pulse wavelength approaching the band-gap. The group index (n(g)) of the L-V(g) region in the Ht-V(g) PC-WG was measured by the cross-correlation method and attained a value above 20. On the other hand, the transmittance of the Ht-V(g) structure recovered approximately 16dB compared to the homogeneous L-V(g) WG having same n(g), 17. This recovery is mainly dominated by the coupling improvement due to the Ht-V(g) structure, around 12dB. These results indicate the effectiveness of the Ht-V(g) structure to use slow light in a PC-WG, which leads to various applications in PC-based optical devices.

Topology optimization has been used to design intersections in two-dimensional photonic crystal slab waveguides. We have experimentally confirmed that the optimized intersection displays high-transmittance with low-crosstalk for the straightforward beam-propagation line.

Antigen-antibody precipitates can be dissolved by the complement cascade (1). The complement-mediated solubilization of immune precipitates requires activation of C3 via the alternative pathway (2) but not participation of the late-acting components, C5 through C9 (1, 3). The classical pathway alone, in the absence of the alternative pathway, is not sufficient to solubilize immune complexes (3).It has recently been demonstrated that the alternative pathway can be assembled from six isolated plasma proteins: C3, factors B and D, properdin, C3b inactivator (C3bINA), x and fllH (4, 5). Cells such as rabbit erythrocytes (6), human lymphoblastoid (Raji) cells (7), and Escherichia coli (8) have been shown to be lysed by the isolated alternative pathway proteins and membrane attack pathway proteins. The question therefore arose as to whether the composite mixture of the isolated alternative pathway proteins is capable of solubilizing immune precipitates. In this communication we report that, indeed, the six isolated alternative pathway proteins can solubilize immune precipitates. In addition, we report on the role of the three regulatory factors (C3bINA, fllH, and properdin) in the solubilization process. Materials and MethodsBuffers. PB: isotonic phosphate-buffered saline, pH 7.4; Mg-GPB: PB containing 0.1% gelatin and 1.2 mM MgC12; EDTA-GPB: PB containing 0.1% gelatin and l0 mM EDTA; DGVB: isotonic veronal buffered saline, pH 7.4, containing 0.1% gelatin, 0.15 mM CaCI2, 0.5 mM MgCI2, and 2.5% dextrose.Immune Precipitates. Egg albumin (grade V) was purchased from Sigma Chemical Company, St. Louis, Mo. Rabbit antiserum to egg albumin was prepared by injecting a rabbit with egg albumin incorporated in complete Freund's adjuvant. IgG antibody to egg albumin was isolated by DEAE-cellulose column chromatograph y25and by affinity chromatography on Sepharose 4B-egg albumin. The IgG was labeled with I by the chloramin T method (9). The specific activity was 5.5 × 106 cpm/p.g protein.Immune complexes were prepared at equivalence with egg albumin and labeled IgG antibody to egg albumin. The mixture was incubated at 37°C for 30 min, then at 4°C overnight. The resulting precipitates were washed three times by centrifugation in the cold and resuspended in Mg-GPB. * Supported, in part, by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, the Governmenl of Japan.~Abbreviations used in this paper: Ab, antibody; C3bINA, C3b inactivator; DGVB, isotonic veronal buffered saline, pH 7.4, containing 0.1% gelatin, 0.15 mM CaCI2, 0.5 mM MgCI2, and 2.5% dextrose; EDTA-GPB, PB containing 0.1% gelatin and 10 mM EDTA; Mg-GPB, PB containing 0.1% gelatin and 1.2 mM MgCI2; PB, isotonic phosphate-buffered saline, pH 7.4; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.J. Ex~,. M r~n.

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