We have shown previously that normal B cells share, with Epstein-Barr virus-transformed and malignant B cells, the ability to activate the alternative pathway (AP) of complement in vitro, resulting in the deposition of C3 fragments on the cell surface. Complement receptor type 2 (CR2, CD21) has been implicated directly as the site for formation of an AP convertase, which provides nascent C3b for deposition at secondary sites on the B-cell surface. In the present study, we have examined C3 fragment deposition in vitro in more detail by (1) assessing the importance of locally generated C3b for the deposition process, (2) investigating whether CR2 is the sole requirement for conferring AP activation capacity on a cell, and (3) determining whether CR2's function, as an AP activator, has different structural requirements from ligand binding. Increasing the availability of native C3, by increasing the serum (NHS) concentration, resulted in enhanced C3 fragment deposition on the B cells, whereas use of factor 1-depleted NHS, which showed massive fluid phase C3 conversion during the incubation, diminished the deposition. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of untreated and hydroxylamine-treated lysates from B cells, after in vitro activation, revealed that the majority of C3 fragments (primarily iC3b and C3dg) had been covalently bound to the cell surface. Transfection of COS cells with wild-type CR2 or a deletion mutant lacking 11 of the molecule's 15 homologous domains, but retaining the ligand-binding site, revealed that expression of intact CR2 conferred a 12-fold increase in AP-activating capacity on these cells, while no increase in AP activity was apparent on cells transfected with the mutant CR2.

Sterols are an essential component of membranes in all eukaryotic cells and the precursor of multiple indispensable cellular metabolites. After endocytotic uptake, sterols are integrated into the lysosomal membrane by the Niemann-Pick type C (NPC) system before redistribution to other membranes. The process is driven by two proteins that, together, compose the NPC system: the lysosomal sterol shuttle protein NPC2 and the membrane protein NPC1 (named NCR1 in fungi), which integrates sterols into the lysosomal membrane. The Saccharomyces cerevisiae NPC system provides a compelling model to study the molecular mechanism of sterol integration into membranes and sterol homeostasis. This review summarizes recent advances in the field, and by interpreting available structural data, we propose a unifying conceptual model for sterol loading, transfer and transport by NPC proteins.

Immunol 1997;46:246-253 Contradictory reports regarding the ability of complement receptor type 2 (CR2,CD21) on normal B cells to activate complement (C 0 ) via the alternative pathway (AP), prompted us to compare the performance of human peripheral blood B cells and the Epstein-Barr virus-positive Burkitt's lymphoma cell line, Raji (a well characterized AP activator) by using flow cytometry. Measured in terms of the membrane deposition of C3 fragments per cell, Raji cells were significantly (6-to 26-fold) more effective as complement activators than were normal B cells. Raji cells were also found to express approximately four to five times as many CR2 as normal B cells. In addition, they distinguished themselves by displaying a greater Ca 2þ -dependent activation, with pooled normal human sera (NHS) as the complement source, and by degrading unprotected C3b fragments from iC3b to C3dg/C3d at a significantly lower rate than the B cells. The Ca 2þ dependency of Raji cell activation was found to be partially a result of classical pathway (CP) triggering by specific antibodies in the NHS, although other triggering mechanisms may also be involved. If the influence of these variations between Raji cells and normal B cells was excluded, by relating deposition of anti-C3d-reactive fragments, during AP activation, to the number of CR2 expressed, the difference in performance between the two cell types was found to be insignificant.

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