Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolyticaIly inactive, fluid-phase C5b-9 complexes, Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3BI, 3Dg, and 2F3), sheep (clones 3Dg and 2F3) and guinea-pig (clone 3D8) neoantigens were identified . Three of four tested clones (3D8, 2F3, IA12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.     

SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells

The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.     

Further glaucolides from south african Vernonia species

The investigation of three South African Vernonia species afforded minute amounts of five new glaucolides, two monoepoxides and three diepoxides. The structures were elucidated by ¹H NMR spectroscopy. The roots of Vernonia sutherlandii contain, in addition to vernonataloide, bergamotene and santalene, minute amounts of the corresponding acetoxy derivatives.     

6-Hydroxytoxol esters and a seco-dammarane derivative from Abrotanella forsterioides

Abrotanella forsterioides afrorded euparin, 6-hydroxytremetone and three 6-hydroxytoxol esters, one of them not being isolated previously. Furthermore a seco-triterpene was isolated. The tribal position of the genus Abrotanella in the Compositae is still an unsolved problem. Morphological investigations suggest that this genus should be transferred from the tribe Anthemideae to the Senecioneae [1,2]. So far two species have been studied chemically; one atforded ent-kaurane derivatives, while both contained euparin and hydroxytremetone [3].     

Umwandlungen der submikroskopischen Chloroplastenstruktur parallel zur Veränderung der stoffwechselphysiologischen Leistung von Chlamydobotrys stellata

Zusammenfassung In Fortsetzung unserer Untersuchungen über den Zusammenhang von Chloroplastenstruktur und-funktion wurde die Veränderung der Chloroplastenstruktur bei der Adaptation photo-heterotroph kultivierter Organismen an photo-autotrophe Ernährungsbedingungen untersucht. Die Bildung von Partitions aus zuvor isolierten Thylakoiden erfolgt parallel zum Anstieg der photosynthetischen CO₂-Assimilation.

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Conformational changes of the submicroscopic chloroplast structure and physiological activity of Chlamydobotrys stellata

Summary Our investigation of the connection between the submicroscopic structure and the function of chloroplasts has been continued. It could be demonstrated that the formation of partitions from isolated thylakoids takes place parallel to the increase of photosynthetic activity.

     

Beziehungen zwischen submikroskopischer Chloroplastenstruktur und Art der Kohlenstoffquelle unter phototrophen Ernährungsbedingungen bei Chlamydobotrys stellata

Zusammenfassung Die Chloroplasten von photo-autotroph (Licht + CO₂) oder photo-heterotroph (Licht + Acetat) gewachsenen Chlamydobotrys stellata werden elektronenmikroskopisch untersucht. Es wird eine nahe Beziehung zwischen der Chloroplasten-Feinstruktur und der Ernährungsart gefunden. Die Thylakoide der Chloroplasten photo-heterotroph kultivierter Algen sind im allgemeinen durch Stroma voneinander getrennt und nur zu wenigen gestapelt. In photo-autotrophen Organismen kommt es durch Bildung einer charakteristischen Faltungsstruktur von Thylakoidmembranen zur Bildung von Grana (Pseudo-Grana).Die Ergebnisse werden im Hinblick auf die Photosynthese und Photoassimilation von Acetat bei Chlamydobotrys stellata diskutiert.

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Relationship between submicroscopic chloroplast structure and type of carbon nutrition of Chlamydobotrys stellata

Summary Chloroplasts from Chlamydobotrys stellata grown photo-autotrophically (light + CO₂) or photo-heterotrophically (light + acetate) have been investigated by means of electronmicroscopy. A close relationship between chloroplast fine structure and type of nutrition could be observed. Thylakoids in chloroplasts from photo-heterotrophically cultivated algae are generally separated from each other by stroma and only few thylakoid packages are present. In photo-autotrophic organisms, however, characteristic folding structure of thylakoid membranes results in the formation of grana (pseudo-grana).These results are discussed in respect to chloroplast function in photosynthesis and photoassimilation of acetate in Chlamydobotrys stellata.

     

Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles.

The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.