Expression of BCL-2, BAX and BAK in the trophoblast layer of the term human placenta: a unique model of apoptosis within a syncytium.

The regulation of apoptosis in the syncytiotrophoblast is of particular interest because this is the only true syncytial epithelium in human cell biology. Nuclei characteristic of apoptotic cells have been localized to this syncytium especially in association with fibrin-containing fibrinoid deposits. The factors responsible for regulating cell death-like features in the trophoblast syncytium are unknown. We tested the hypothesis that fibrin was required for trophoblast apoptosis. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP end-labelling) staining to detect DNA fragmentation typical of apoptosis was performed in term human placentae revealing labelled nuclei associated with fibrin-type fibrinoid, as well as labelled nuclei in discrete areas of syncytiotrophoblast without fibrin. We also hypothesized that members of the BCL-2 family of apoptosis-associated proteins contribute to the regulation of syncytiotrophoblast apoptosis. To identify members of this protein family that might regulate trophoblast apoptosis, we assessed expression of three important members of the bcl-2 gene family. We used immunohistochemistry with monoclonal antisera against human BCL-2 and polyclonal antisera against human BAX and BAK to study paraffin-embedded sections of human term placentae (n=5) from uncomplicated pregnancies. The anti-apoptotic BCL-2 protein was expressed throughout the syncytium of normal villi with much less staining in cytotrophoblast. Staining was also seen adjacent to fibrin deposits and in syncytium overlying fibrin deposits. Expression of the pro-apoptotic BAX protein was undetectable in the syncytiotrophoblast, was expressed in rare cytotrophoblast and was prominent in connective tissue and perivascular cells within the villous core. Localization of a second pro-apoptotic protein, BAK, revealed immunoreactivity in isolated areas of intact syncytium of normal villi. Additionally, fibrin deposits were associated with intense BAK staining in both syncytiotrophoblast and cytotrophoblast. From these data, we speculate that modulation of BAK expression is one factor regulating apoptosis in human trophoblast.

The varicella-zoster virus origin-binding protein can substitute for the herpes simplex virus origin-binding protein in a transient origin-dependent DNA replication assay in insect cells.

We isolated two recombinant baculoviruses each of which expresses a varicella-zoster virus (VZV) homolog of one of the seven herpes simplex virus type 1 (HSV-1) genes required for DNA replication. We performed transient origin-dependent DNA replication assays in insect cells in which we substituted a baculovirus which expresses a VZV protein for a baculovirus which expresses its HSV homolog. VZV gene 51 protein was found to be able to support origin-dependent DNA synthesis when it was substituted for UL9, the HSV-1 origin-binding protein (OBP). This occurred whether an HSV-1 or a VZV origin-containing plasmid was used in the assay. These results suggest that VZV gene 51 protein is able to interact with the HSV replication machinery, and in light of the extensive structural divergence of these proteins, it suggests that initiation of VZV and HSV-1 DNA synthesis may involve a limited number of interactions between the OBP and other replication factors. Substitution of infected-cell protein 8 (ICP8), the major single-stranded DNA-binding protein of HSV-1, with VZV gene 29 protein, however, did not result in amplification of plasmids containing either an HSV-1 or a VZV origin. In the absence of ICP8, addition of both VZV gene 51 protein and gene 29 protein was also negative for origin-dependent replication whether or not UL9 was present. Although demonstration that our baculovirus-expressed VZV gene 29 protein is functional for DNA replication will await development of a VZV replication system, our results suggest that VZV gene 29 protein is unable to interact functionally with one or more of the HSV replication proteins. This approach should contribute to efforts to define the interactions among the alphaherpesvirus DNA replication proteins.

Analysis of C3b/C3d binding sites and factor I cofactor regions within mouse complement receptors 1 and 2.

Human and murine CR1 and CR2 are defined as evolutionary homologues on the basis of their in vitro activities and a shared structural motif known as a short consensus repeat (SCR). To identify additional similarities between the two species, we analyzed the functional domains within the mouse receptors by constructing mouse-human chimeric cDNAs in which the C3 binding site of human CR2 has been replaced by different regions within the first eight SCRs of mouse CR1. Rosette analysis of cells expressing chimeric proteins, with erythrocytes bearing different mouse C3 fragments, coupled with rosette inhibition studies using specific anti-mouse CR1/CR2 mAbs reveal a weak C3b binding site within SCRs 1 and 2 of mouse CR1. There is no independent C3b interacting domain within SCRs 3 to 6, but their presence enhances C3 binding. A molecule that contains only the first six SCRs of mouse CR1 also binds C3b, but with less efficiency. There is no C3d binding area within the first six SCRs, but our data confirms previous studies indicating an additional C3b/C3d binding region within SCRs 7 and 8 of mouse CR1 (SCRs 1-2 of mouse CR2). The presence of SCRs 1 to 4 is required for C3 cofactor activity. 8C12, a mAb which blocks C3b erythrocyte rosette binding and the C3 cofactor activity of mouse CR1, binds only to chimeras containing SCRs 3 to 4.(ABSTRACT TRUNCATED AT 250 WORDS)